A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum-associated degradation.

The endoplasmic reticulum-associated degradation (ERAD) pathway mediates the endoplasmic reticulum-to-cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K-resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.

New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.

Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.

Cuidar da saúde do vizinho: atuação do antropólogo Charles Wagley no Serviço Especial de Saúde Pública* / Caring for the health of your neighbor: the work of anthropologist Charles Wagley with the Serviço Especial de Saúde Pública

Recupera a atuação do antropólogo Charles Wagley como alto funcionário do Serviço Especial de Saúde Pública, programa de cooperação estabelecido entre EUA e Brasil na Segunda Guerra Mundial. Convocado a colaborar nos esforços de guerra, atuou na política de migração do Programa da Borracha. À luz dessa experiência de intervenção, do contexto marcado pela promoção do desenvolvimento e por questões então prementes no campo da antropologia, este estudo propõe-se retomar a obra Uma comunidade amazônica. Trata-se de discutir o estudo de comunidade conduzido na localidade amazônica que Wagley conheceu ainda durante as missões do Serviço e cuja realidade considerou ilustrativa de uma região subdesenvolvida, levando-o a refletir sobre mudança social e o papel das ciências.

The article focuses on the work of Charles Wagley as a top staff member with Serviço Especial de Saúde Pública (Special Public Health Service), a US-Brazil cooperation program established during World War II. Taking into consideration Wagley’s experience with migration policy under Brazil’s Rubber Program, as well as the context of development promotion and the issues then on the anthropological agenda, the article explores Wagley’s community study of the Amazon town he visited while on SESP missions, published in the book Uma comunidade amazônica (Amazon Town). Encountering a reality that he believed emblematic of underdevelopment, Wagley was led to reflect on social change and the role of science.

Methotrexate analogues display enhanced inhibition of TNF-α production in whole blood from RA patients.

OBJECTIVES: Although methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA), patients experience clinical resistance to MTX upon prolonged treatment. We explored whether new-generation antifolates elicit superior anti-inflammatory properties when compared to MTX, based on their capacity to inhibit tumour necrosis factor (TNF)-α production. METHOD: T cells in whole blood from 18 RA patients (including MTX-naïve, MTX- responsive, and MTX non-responsive patients) and seven healthy volunteers were stimulated with αCD3/αCD28 antibodies and incubated ex vivo for 72 h with MTX and eight novel antifolate drugs with potentially favourable biochemical and pharmacological properties. Drug concentrations exerting 50% inhibition (IC-50) of TNF-α production (by enzyme-linked immunosorbent assay, ELISA) were determined as an estimate for their anti-inflammatory capacity. In addition, induction of T-cell apoptosis was evaluated by flow cytometry. RESULTS: The new-generation antifolates PT523, PT644, raltitrexed, and GW1843 proved to be potent inhibitors of TNF-α production in activated T cells from all three groups of RA patients and from healthy volunteers. Based on IC-50 values, these antifolates were up to 10.3 times more potent than MTX. The anti-inflammatory effects were observed at drug concentrations that provoked suppression of T-cell activation and induction of apoptosis in 20-40% of activated T cells. CONCLUSION: In an ex-vivo setting, novel antifolates elicited marked inhibition of TNF-α production in activated T cells from RA patients. Further clinical evaluation is warranted to investigate whether a low dosage of these antifolates can elicit immunosuppressive effects equivalent to MTX, and whether they are superior to MTX in patients who fail to respond to MTX.

Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer.

The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC50 < 50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria.

Impairments in antifolate transport are common in retinoblastoma tumor samples.

BACKGROUND: Many patients with retinoblastoma have a genetic predisposition to cancer and external beam radiation therapy and alkylating agent chemotherapy may increase their risk of secondary malignancy. Identification of effective chemotherapy agents for retinoblastoma that are not associated with an elevated risk of secondary malignancy would be beneficial. PROCEDURE: Twenty-six specimens of fresh retinoblastoma tumor cells were studied in vitro with a PT430 competitive displacement assay. Differential displacement of the PT430 by methotrexate and not trimetrexate was considered indicative of a defect in reduced folate carrier (RFC)-mediated transport. Elevations in the accumulation of PT430 were considered indicative of dihydrofolate reductase (DHFR) amplification. RESULTS: In 9 of the 26 (35%) samples, displacement by methotrexate was less than half the displacement by trimetrexate indicative of a defect in the RFC. In 5 of the 26 (19%) samples, trimetrexate did not displace the PT430. In 7 of 26 (27%) samples, the peak PT430 accumulation was suggestive of DHFR overexpression. Overall 9 of 26 (35%) samples had no evidence of a transport defect or DHFR overexpression and would be anticipated to be potentially sensitive to methotrexate. In 15 of the 26 (58%), no defects existed in trimetrexate displacement or DHFR overexpression and would be anticipated to be potentially sensitive to trimetrexate. CONCLUSION: These results would support consideration of a phase II study to determine the effectiveness of trimetrexate for recurrent intra-ocular retinoblastoma.

Complete regression of large solid tumors using engineered drug-resistant hematopoietic cells and anti-CD137 immunotherapy.

Combining chemotherapy and immunotherapy is problematic because chemotherapy can ablate the immune responses initiated by modulators of the immune system. We hypothesized that protection of immunocompetent cells from the toxic effects of chemotherapy, using drug resistance gene therapy strategies, would allow the combined use of chemotherapy and immunotherapy. In wild-type mice, the antitumor effectiveness of an immunotherapy regimen employing an agonistic anti-CD137 antibody is diminished with escalating doses of the antifolate trimetrexate (TMTX). Using retroviral gene transfer of a mutant form of dihydrofolate reductase (L22Y-DHFR), hematopoietic stem cells were genetically engineered to withstand the toxic effects of TMTX. Mice transplanted with L22Y-DHFR-modified bone marrow were then challenged with AG104 sarcoma cells and treated with TMTX only, anti-CD137 only, or a combination of chemotherapy and immunotherapy. Although tumor burden was transiently decreased during TMTX administration, no mice treated with TMTX alone survived the tumor challenge, whereas approximately 40% of transplanted mice treated with anti-CD137 alone survived. However, 100% of mice survived with complete tumor regression after transplantation with L22Y-DHFR-transduced bone marrow followed by combined treatment with TMTX and anti-CD137. In addition, adoptive transfer of splenocytes from cured mice extended the survival of tumor- bearing animals by approximately 3 weeks compared with controls. Therefore, protection of the hematopoietic system can allow for the combined administration of chemotherapy and immunotherapy, which results in complete tumor clearance.

Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates.

Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.

Superior depletion of alloreactive T cells from peripheral blood stem cell and umbilical cord blood grafts by the combined use of trimetrexate and interleukin-2 immunotoxin.

Acute graft-versus-host disease, a major obstacle to the overall success of allogeneic hematopoietic stem cell transplantation, is primarily induced by a subset of donor T cells. Most strategies to prevent acute graft-versus-host disease target all T cells regardless of their specificity, and this leads to prolonged posttransplantation immunodeficiency. Selective depletion of alloreactive T cells could spare protective immunity and facilitate engraftment and graft-versus-leukemia effects. Recently described depletion strategies target activation markers such as CD25 that are expressed by alloreactive T cells. However, incomplete depletion may occur when a single surface epitope or pathway of apoptosis is targeted that may not be fully and concurrently expressed among all alloreactive cells. We now report on a novel strategy effective in both cord blood and peripheral blood stem cell alloreactive T cells that simultaneously induces 2 independent pathways of apoptosis after stimulation by recipient dendritic cells or Epstein-Barr virus-transformed B cells. First, we demonstrate that the folate antagonist trimetrexate selectively depletes proliferating alloreactive precursors in vitro in a dose- and time-dependent manner. Similarly, a second agent, denileukin diftitox, kills activated alloreactive T cells expressing CD25. Most importantly, these 2 agents can exert their effects in concert with superior efficacy while sparing resting bystander T cells, which remain available to mount antimicrobial or third-party responses.

Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, approximately 45 nmol/L) than methotrexate (MTX; Kt, approximately 1 micromol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were approximately 400-600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 micromol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 micromol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (approximately 1.2 micromol/L) or the Ki for folic acid (approximately 130 micromol/L) or PT523 ( approximately 0.2 micromol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl- (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

p14ARF expression increases dihydrofolate reductase degradation and paradoxically results in resistance to folate antagonists in cells with nonfunctional p53.

The p14(ARF) protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G(1) and G(2) arrest of cells in the cell cycle. Although much is known about the function of p14(ARF) in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14(ARF) and harboring a defective p53 pathway were stably transfected with p14(ARF) cDNA under the tight control of a doxycycline-inducible promoter. Induction of p14(ARF) caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14(ARF) on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14(ARF) augments proteasomal degradation of the protein. Surprisingly, induction of p14(ARF) increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14(ARF) induction increases the reliance of these cells on thymidine salvage.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors.

One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example.

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.

Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin.

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.

Trimetrexate inhibits progression of the murine 32Dp210 model of chronic myeloid leukemia in animals expressing drug-resistant dihydrofolate reductase.

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). Administration of the maximum tolerated dose of trimetrexate (TMTX) with the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose-5'-monophosphate (NBMPR-P) inhibited 32Dp210 tumor progression in mice engrafted with transgenic tyrosine-22 DHFR marrow and improved survival of tumor-bearing animals as long as drug administration was continued. NBMPR-P coadministration was necessary for maximal tumor inhibition, as administration of TMTX alone delayed but did not prevent tumor progression. The chemoprotection afforded by engraftment with transgenic tyrosine-22 DHFR marrow was necessary for effective chemotherapy, as normal mice lacking transgenic marrow could not tolerate the higher TMTX dose (60 mg/kg/day) administered to mice with transgenic marrow, and the decreased dose of TMTX with NBMPR-P tolerated by normal tumor-bearing animals did not inhibit tumor progression or improve animal survival. We conclude that TMTX with NBMPR-P inhibits tumor progression in the 32Dp210 model of CML in animals engrafted with drug-resistant tyrosine-22 DHFR transgenic marrow, and that based on this model the introduction of a drug-resistant DHFR gene into marrow combined with TMTX and NBMPR-P administration may provide an effective treatment for CML.

Severe folate restriction results in depletion of and alteration in the composition of the intracellular folate pool, moderate sensitization to methotrexate and trimetrexate, upregulation of endogenous DHFR activity, and overexpression of metallothionein II and folate receptor alpha that, upon folate repletion, confer drug resistance to CHL cells.

DC-3F/FA3 cells (FA3) were derived from antifolate-sensitive CHL cells by selection for growth in folate-free media containing 15 pM [6S]-5CHOFH4. These cells undergo a 30-fold decrease in intracellular folates, overexpress folate receptor alpha (FR alpha) and metallothionein II, and display increased sensitivity to the dihydrofolate reductase (DHFR) targeted anti-folates methotrexate (MTX) and trimetrexate (TMTX), which can be attributed primarily to the folate pool status. Upon folate repletion by growth in 15 nM [6S]-5CHOFH4, they display a 5- and 10-fold increase in resistance to both drugs, respectively, even though folate pools are restored by only 43%. Enforced overexpression of FR alpha in transfectants cultured in nanomolar folate did not confer resistance to MTX but did support a modest 2-fold increase in resistance to TMTX. Enforced overexpression of MTII had a similar effect, but when both were overexpressed together no increase in resistance beyond that conferred by each one separately was noted, suggesting that both confer resistance to TMTX through a common downstream mechanism. Analysis of three independent low folate selected clones, FA3, FA7, and FA14, showed that each had a 5- to 6-fold increase in DHFR activity accompanied by a similar increase in DHFR protein level. However, no differences were detected in the DHFR gene copy number or in the steady-state amount of DHFR mRNA, suggesting that a posttranscriptional mechanism was responsible for the increase in DHFR expression.

The murine-reduced folate carrier gene can act as a selectable marker and a suicide gene in hematopoietic cells in vivo.

Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.

Trimetrexate in relapsed T-cell lymphoma with skin involvement.

PURPOSE: Methotrexate (MTX) is active against lymphomas, but transport or polyglutamylation mutations confer MTX resistance. Because trimetrexate (TMTX) enters cells by passive diffusion and is not polyglutamylated, its activity in relapsed T-cell lymphoma was investigated. PATIENTS AND METHODS: Eligible patients had histologically confirmed relapsed T-cell lymphoma involving the skin, had received more than one previous regimen, were older than 16 years, had normal organ function, and had no CNS disease or serious infections, including human immunodeficiency virus. TMTX (200 mg/m(2)) was given intravenously every 14 days without topical or systemic corticosteroids. Patients who responded received up to 12 doses. RESULTS: Twenty patients were assessable for response. Median age was 59 years (range, 45 to 87 years); 13 patients were men. Three patients had anaplastic large-cell lymphoma, 15 had mycosis fungoides or Sézary syndrome (14 with large-cell transformation), and two had peripheral T-cell lymphoma. Serum lactate dehydrogenase was high in 35%, and beta-2 microglobulin was more than 3.0 mg/L in 35% of patients. The median number of previous regimens was three (range, two to 15) and included MTX in five patients. Disease was refractory to the regimen immediately preceding TMTX in 85% of patients. Responses were complete in one and partial in eight patients (overall response rate, 45%). Two of five patients previously treated with MTX responded. Grade 3 or 4 mucositis was observed after 4%, infection after 3%, neutropenic fever after 6%, neutrophils less than 100/microL after 4%, and platelets less than 10,000/microL after 3% of TMTX doses. CONCLUSION: TMTX is active with acceptable toxicity in this population and merits further investigation.

Methylthioadenosine phosphorylase as target for chemoselective treatment of T-cell acute lymphoblastic leukemic cells.

We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.

Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: a method to modulate gene expression.

Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drug-mediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.