Effects of maitake (Grifola frondosa) D-Fraction on the carcinoma angiogenesis

We have reported that D-Fraction extracted from maitake (Grifola frondosa), activates immune competent cells, and indicates anti-tumor activities. The D-Fraction was observed to induce angiogenesis in vivo and to enhance the proliferation capability and migration capability of human vascular endothelial cell in vitro. The D-Fraction also increased plasma vascular endothelial growth factor (VEGF) concentration significantly. Also VEGF and TNF-alpha production by the activated peritoneal macrophages were enhanced. These results suggest that the anti-tumor activity of the D-Fraction is not only associated with the activation of the immuno-competent cells but also possibly related to the carcinoma angiogenesis induction.     

Nitric oxide-mediated antitumor activity induced by the extract from Grifola frondosa (Maitake mushroom) in a macrophage cell line, RAW264.7

We have investigated D-fraction (MDF) extracted from Grifola frondosa (Maitake mushroom) on the inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 (RAW) cells, a murine monocyte/macrophage cell line, with special reference to antitumor activity of MDF against human hepatoma-derived huH-1 cells. MDF could induce iNOS mRNA expression in RAW cells in a dose range of more than 30 microg/ml, but the effect of 10 microg/ml of MDF was negligible. The iNOS mRNA expression induced by 100 microg/ml of MDF was 6 hrs later, but lasted for a longer time than that of lipopolysaccharide (LPS), a representative iNOS inducer. Although iNOS mRNA levels in MDF-stimulated cells were almost equal to LPS-stimulated cells at the peak time, the cumulative amount of nitrite was only about 50% compared with that of LPS-treated cells. When huH-I cells were cultured in MDF containing media in a 24-well plate with inserted porous bottom in the presence or absence of RAW cells, the viability of huH-1 cells decreased significantly only in the presence of RAW cells in MDF dose-dependent manner. This antitumor activity of RAW cells in the presence of MDF was abolished or attenuated by the addition of L-NAME, a NOS inhibitor, confirming that this phenomenon is due to iNOS-mediated NO production by RAW cells, but not direct cytotoxic activity of MDF against huH-1 cells. These data suggest that MDF is a novel inducer for iNOS which contributes at least in part to antitumor activity of MDF.     

耐地塞米松B细胞淋巴瘤细胞逃逸同种异体NK细胞的杀伤及其机制

目的:观察地塞米松耐药的人B细胞淋巴瘤细胞系对NK细胞杀伤敏感性的变化,并探讨其作用机制。方法:20μg/ml地塞米松(dexamethasone,DXM)诱导B细胞淋巴瘤细胞系SU-DHL-4(简称SU细胞)发生耐药,建立多药耐药细胞系SU/DXM。流式细胞术分选健康人外周血NK细胞,流式细胞术检测效靶比20∶1时,NK细胞对SU和SU/DXM细胞的杀伤效应。实时定量PCR检测SU和SU/DXM细胞表面NK细胞活化性受体(soluble NK group 2 member D,NKG2D)配体基因[可溶性MHCⅠ类分子相关A/B(MHC classⅠchain-related molecules A/B,MICA/B)及人UL16结合蛋白(UL16 binding protein,ULBP)1、2、3]的表达。结果:成功建立多药耐药细胞系SU/DXM。与SU细胞相比,SU/DXM细胞对NK细胞杀伤的敏感性明显下降[SU细胞为(11.38±3.51)%,SU/DXM细胞为(3.57±4.22)%,P<0.05],细胞表面NKG2D配体基因MICA、MICB、ULBP2 mRNA表达量降低(SU细胞分别为1.014±0.121、1.009±0.092、0.993±0.108,SU/DXM细胞分别为0.017±0.006、0.682±0.063、0.773±0.066,P<0.05或P<0.01)。结论:地塞米松能诱导B细胞淋巴瘤SU细胞发生多药耐药,多药耐药SU/DXM细胞能够抵抗NK细胞的杀伤,其机制可能与NKG2D配体基因表达量下降有关。     

Enhancement of cytotoxicity by hyperthermia after a long-term culture with 5-fluorouracil in transformed cells.

The cytotoxic effect of 5-FU (5-fluorouracil) was demonstrated to be enhanced by hyperthermia after treatment with 5-FU at even a comparatively low dose over fairly long periods. The cytotoxic effect of the combined treatment with 42 degrees C-hyperthermia and 1 microgram/ml 5-FU for 48 hrs, the cytotoxic effect of 42 degrees C-hyperthermia and 5 micrograms/ml 5-FU for 24 hrs, and the cytotoxic effect of 43 degrees C-hyperthermia and 1 and 5 micrograms/ml 5-FU for 8 hrs were studied. The maximally enhanced rate of 42 degrees C-hyperthermia after 5-FU treatment for 96 hrs was 48% after a 1 microgram/ml 5-FU treatment and 150% after 45 hrs with the 5 micrograms/ml 5-FU treatment. The maximally enhanced rate of 43 degrees C-hyperthermia after 5-FU treatment was 170% after 45 hrs with 1 microgram/ml 5-FU treatment and 180% after 24 hrs with 5 mg/ml 5-FU treatment. When V-79 cells were treated at the same temperature, the maximally enhanced rate with 1 microgram/ml 5-FU was almost equal to that of 5 micrograms/ml. Moreover, when each maximally enhanced rate was equalized, each concentration of FU (RNA)/RNA practically became equal, i.e., when the maximally enhanced rates were approximately 150 and 170-180%, FU (RNA)/RNA concentrations were about 40 and 15 ng/mg RNA, respectively. We thus concluded that FU (RNA)/RNA concentration might play an important role as an indicator of the effect of the combined treatment of 5-FU and hyperthermia.     

Enhancement of etoposide-induced cytotoxicity by cyclosporin A

Summary Following the clinical observation of enhanced antineoplastic action of etoposide in the presence of cyclosporin A (CyA), we investigated this drug interaction in several in vitro and in vivo tumor systems. Macromolecular DNA damage induced by etoposide at drug levels comparable to plasma AUC values achieved in patients was increased not only in leukemic peripheral blood cells from patients but also in mononuclear peripheral blood cells from a healthy donor. Intracellular retention of radioactivity from ³H-etoposide was increased by a factor of 1.5 at the most in the presence of CyA. The cytotoxicity of etoposide and adriamycin to L 1210 leukemic cells was clearly enhanced, whereas CyA had no effect on the action of cisplatin or ionizing irradiation. At CyA blood levels not exceeding 1.44 g/ml, increased tumor inhibition of etoposide was observed in a human embryonal cancer xenograft, but there was also higher lethality in normal mice. We conclude from our own data and from other recent findings that with respect to chemosensitization the effects of CyA resemble those of calcium channel blockers or anticalmodulin agents. In contrast to calcium channel blockers, however, adequate plasma levels of CyA can well be achieved in patients.Supported by BDMFT (PTB 8315)Supported by SFB 102 (Deutsche Forschungsgemeinschaft)     

Enhancement by caffeine of neocarzinostatin cytotoxicity in murine leukemia L1210 cells.

Posttreatment incubation with nontoxic doses of caffeine resulted in enhancement of cell lethality and inhibition of cell growth in L1210 mouse leukemia cells which had been exposed to a protein antibiotic, neocarzinostatin. In addition, caffeine treatment appeared to inhibit the eventual maturation of newly synthesized DNA in L1210 cells following exposure to this antibiotic. These results, indicating the existence of caffeine-sensitive repair in L1210 leukemia cells treated with neocarzinostatin, provide further evidence for DNA damage as a mechanism of the cytocidal action of the antibiotic.     

Enhancement of nitrocaphane cytotoxicity by hyperthermia in vitro

HEp-2 cells were treated with hyperthermia (39–44°C) and nitrocaphane (NC) at various time intervals. A 1 h exposure to 39°C and 41 °C was non-lethal to cells, but it did potentiate the cell killing of NC (1.0 μg/ml). It was further shown that the sequence between heat and the drug can affect cell survival. Cell killing effect was decreased when heat was given before or after administration of drug. In contrast the simultaneous administration of these two modalities was synergistic. Maximal thermotolerance of HEp-2 cells was developed using an 8-h interval at 37 °C between the cell exposures to two equal thermal doses (44°C, 30 min). HEp-2 cells became thermotolerant when preheated for 30 min at 44°C followed by a 10-h interval at 37°C. The thermotolerant cells showed resistance to subsequent heat at 44°C (D₀=2.26 h, control D₀=0.38 h), to subsequent NC treatments, and to heat combined with NC. However, in the thermotolerant cells, cytotoxicity of NC was still enhanced by hyperthermia.     

Enhancement of vinblastine-induced cytotoxicity by lysolecithin and phosphatidylinositol

Vinblastine inhibited the growth of cultured KB cells 3 days after drug treatment by 55% and 67% at 2.7 ng/ml and 3.5 ng/ml of the medium, respectively. Lysolecithin and phosphatidylinositol showed only a marginal inhibitory effect on the growth of KB cells at respective concentrations of 35–125 μg/ml and 50–150 μg/ml of the medium. Lysolecithin, however, enhanced the cytotoxicity of vinblastine. Depending upon the concentrations of lysolecithin (35–125 μg/ml), the growth of KB cells was inhibited by 60–91% and 86–98% at respective vinblastine concentrations of 2.7 ng/ml and 3.5 ng/ml. Enhancement of vinblastine-induced cytotoxicity also occurred similarly for phosphatidylinositol. The mechanism could be explained partly by an elevated amount of intracellular vinblastine. Other possible mechanisms can only be speculated.     

Enhancement of nitrocaphane cytotoxicity by hyperthermia in vitro

HEp-2 cells were treated with hyperthermia (39-44 degrees C) and nitrocaphane (NC) at various time intervals. A 1 h exposure to 39 degrees C and 41 degrees C was non-lethal to cells, but it did potentiate the cell killing of NC (1.0 micrograms/ml). It was further shown that the sequence between heat and the drug can affect cell survival. Cell killing effect was decreased when heat was given before or after administration of drug. In contrast the simultaneous administration of these two modalities was synergistic. Maximal thermotolerance of HEp-2 cells was developed using an 8-h interval at 37 degrees C between the cell exposures to two equal thermal doses (44 degrees C, 30 min). HEp-2 cells became thermotolerant when preheated for 30 min at 44 degrees C followed by a 10-h interval at 37 degrees C. The thermotolerant cells showed resistance to subsequent heat at 44 degrees C (D0 = 2.26 h, control D0 = 0.38 h), to subsequent NC treatment, and to heat combined with NC. However, in the thermotolerant cells, cytotoxicity of NC was still enhanced by hyperthermia.     

Enhancement of human natural cell-mediated cytotoxicity by interferon

The effect of exogenous Namalva interferon (IF) on the natural killer (NK) cell activity of human blood lymphocytes was examined against 5 target cell lines (K562, CCRF/CEM, Molt 4, Raji and Bri8) using the 51Cr-release assay. Addition of IF to the test significantly increased the cytotoxicity, though not as much as when effector cells were treated with IF before the test. Augmentation of cytotoxicity was evident after only 1 h pretreatment and was maximal by 6 h. The rate of lysis of susceptible targets by IF-treated effectors markedly exceeded that by their untreated counterparts. Separation of lymphocyte subpopulations (by SRBC-rosette sedimentation and nylon-fibre column filtration) demonstrated that the activities of IF-stimulated and unstimulated cells were similarly distributed, suggesting that the major effect of IF is enhancement of the activity of pre-existing NK cells rather than generation of new populations of effectors. Target cell lines with high and low susceptibility to NK cells showed increased cytotoxicity by IF-treated effector cells. These findings may be relevant to the current discussion of the role of NK cells in immunosurveillance against neoplasia.     

Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism

Regulation of innate immunity may be an effective means of cancer control. Delaying cancer onset is regarded as an important mode of action in cancer prevention. We have been investigating the chemopreventive mechanisms of Lactobacillus casei Shirota (LcS), a probiotic strain. In this study, we evaluated the effect of LcS on tumor onset and the involvement of natural killer (NK) cells using a 3-methylcholanthrene-induced carcinogenesis model. C3H/HeN mice were divided into three groups, according to treatment: vehicle-treated, treated with vehicle only; control, 3-methylcholanthrene treated; LcS, 3-methylcholanthrene and LcS treated. 3-Methylcholanthrene was injected intradermally at 7 weeks of age. LcS was mixed into the diet (0.05%, w/w), which the mice were fed from the day of 3-methylcholanthrene injection onward. Tumor incidence was observed weekly. Profiles of splenic NK cells, in vitro cytotoxicity and the proportion, in the early stage of carcinogenesis were analyzed at 5 weeks after the injection. The tumor suppressive effect of LcS was also evaluated in a beige mouse model that is genetically deficient in NK cells. LcS delayed tumor onset and reduced tumor incidence in the results with C3H/HeN mice (P< 0.05). More specifically, tumor incidence in the control group was 33% at 6 weeks after the injection and 83% at 11 weeks as opposed to 0 and 42%, respectively, in the LcS group. NK cell cytotoxicity was significantly higher than in the control group, and the number of NK cells also increased in the LcS group of C3H/HeN mice. However, LcS failed to suppress tumorigenesis in the beige mouse. These findings suggest that enhancement of the cytotoxicity of NK cells by LcS delays tumor onset.     

Direct RIG-I activation in human NK cells induces TRAIL-dependent cytotoxicity toward autologous melanoma cells

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5′-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy.     

Enhancement of cisplatin cytotoxicity by terbium in cisplatin-resistant MDA/CH human breast cancer cells

Purpose: The development of cisplatin resistance is a major problem in the treatment of cancer patients with cisplatin chemotherapy. The membrane binding of terbium (Tb³⁺) has been shown to increase the cellular accumulation of cisplatin in breast cancer cells. Therefore, the ability of Tb³⁺ to modulate the cytotoxicity of cisplatin was investigated in cisplatin-sensitive (MDA) and cisplatin-resistant (MDA/CH) MDA-MB-231 human breast cancer cells. Methods: The cytotoxic parameters of cisplatin were determined using live cell microfluorometry and median effect analysis. Results: MDA/CH cells (IC₅₀ = 142 ± 9 μM) were found to be approximately 3.3-fold more resistant to cisplatin than MDA cells (IC₅₀ = 43.5 ± 3.0 μM). In both cell lines, the IC₅₀ value for cisplatin was reduced two-fold in the presence of 80 μM Tb³⁺, thus indicating that the cytotoxicity of cisplatin is increased by Tb³⁺. The cytotoxic activity of cisplatin alone was observed to be 5.7 and 1.6 times more potent than that of Tb³⁺ alone in MDA and MDA/CH cells, respectively. Combination index analyses revealed that the interaction between cisplatin and Tb³⁺ was only synergistic at very low indices of cell death in MDA cells. However, in MDA/CH cells, the two drugs were synergistic up to intermediate levels of cell death. Conclusions: Our results suggest that the enhancement of cisplatin cytotoxicity by Tb³⁺ is more effective in cisplatin-resistant MDA/CH cells than in cisplatin-sensitive MDA cells. Therefore, terbium is potentially useful in cisplatin combination therapy for breast cancer patients, especially for those patients who have developed resistance to the drug. Received: 16 December 1998 / Accepted: 19 January 1999     

Enhancement of adriamycin cytotoxicity in sensitive and resistant sublines of human tumor cells by calcium antagonists

The effect of the calcium antagonists cepharanthine and verapamil on adriamycin-induced cytotoxicity against sensitive (K 562 and Ov 2780) and resistant (K 562/ADM and AD 10) sublines of human tumor cells was evaluated. Nontoxic concentrations of cepharanthine moderately enhanced adriamycin cytotoxicity against sensitive sublines (2.1-2.5 fold). A significant enhancement (13-26 fold) of drug cytotoxicity was observed when resistant cells were treated with a combination of cepharanthine and adriamycin. The calcium influx blocker verapamil (used for comparison) also enhanced adriamycin cytotoxicity, although to a lesser extent. The fact that enhancement was 6-10 fold greater in resistant then in sensitive cells, as well as the loss of biphasic properties of adriamycin on dose-response curves after combined treatment, indicate that cepharanthine may play a role in overcoming drug resistance in some tumor cells.     

Enhancement of 5-fluorouracil cytotoxicity on human colon cancer cells by retrovirus-mediated interferon-alpha gene transfer.

Gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Several reports documented the clinical effects of interferon-alpha (IFN-alpha) in management of patients with advanced colorectal carcinoma. We report for the first time, the successful transduction of human IFN-alpha gene into colon cancer cells, COLO 201 using a replication-defective retroviral vector. Retrovirus-containing supernatant from PA 317 packaging cells was used to infect colon cancer cells, COLO 201 and NIH 3T3 cells. Transient infection showed that cell proliferation and cell viability were significantly suppressed in colon cancer cells transduced with IFN-alpha gene. Moreover, IFN-alpha-transduced cells acquired less resistance to 5-FU induced apoptosis. These data demonstrate that IFN-alpha gene transfer may have a clinical application and can be combined with chemotherapy for treatment of advanced colorectal cancer.     

Enhancement of mitomycin C cytotoxicity to hypoxic tumor cells by dicoumarol in vivo and in vitro

Previous work by our laboratories demonstrated that dicoumarol can increase the enzymatic activation of mitomycin C (MC) to alkylating species by tumor cell sonicates under hypoxic conditions. To determine whether this increased generation of reactive metabolites would result in increased cytotoxicity, we examined the effect of this combination on the viability of EMT6 cells treated in vitro under hypoxic and oxygenated conditions. Dicoumarol increased the cytotoxicity of MC to these neoplastic cells under hypoxic conditions and decreased the toxicity of the antibiotic to aerobic cells. These findings suggested that dicoumarol might enhance the toxicity of MC to the hypoxic cells of solid tumors, without increasing the toxic side effects of the antibiotic to the host. Treatment of EMT6 tumor-bearing animals with both dicoumarol and MC significantly decreased the survival of the radioresistant hypoxic tumor cells from that obtained with MC alone. In contrast, the leukopenia produced by the antibiotic was not exacerbated by the addition of dicoumarol. These results suggest that a treatment regimen combining dicoumarol and MC might be a useful adjunct to radiation therapy for the eradication of the radioresistant hypoxic cells in solid tumors.     

A comparison of the NK cell cytotoxicity with effects of TNF-alpha against K-562 cells, determined by LDH release assay.

Effects of r h TNF-alpha as a single cytotoxic mediator against K-562 cells was examined by LDH release and compared with NK cell cytotoxicity. The mean values of the percentage of LDH release (x = 6.25 +/- 3.68%, for ten individual experiments) from K-562 cells cultured for 2 h with r h TNF-alpha 100 U/ml of culture medium did not give significant difference in comparison with mean values of percentage LDH release (x = 6.43 +/- 2.97%, for 37 individual experiments) from K-562 cells which were cultured without r h TNF-alpha (Student's t-test, P > 0.05). The results also showed, that in the presence of increasing concentrations of r h TNF-alpha there was no significant increase of LDH release through the cell membrane in these short term incubations. However, significant difference in LDH release from K-562 cells was found after 6 h between cultures treated for 30 min with or without r h TNF-alpha (Mann-Whitney test, P < 0.05). Since TNF-alpha alone shows a lower degree of K-562 cell membrane damage than NK effectors, this suggested that TNF-alpha is neither an only nor a major mediator of cell destruction, based on determination of LDH release.     

Prophylactic IL-12 treatment reduces postoperative metastasis: mediation by increased numbers but not cytotoxicity of NK cells

Despite a promising potential, interleukin-12 immunotherapy has yielded limited clinical success while causing perilous toxicities. Here we study a context in which IL-12 may prove clinically beneficial—the removal of the primary tumor, when cell-mediated immunity (CMI) may eradicate minimal residual disease (MRD), but is inhibited by postoperative immunosuppression, potentially leading to enhanced malignant progression. F344 rats were preoperatively treated with IL-12 and inoculated postoperatively with syngeneic MADB106 tumor cells. An optimal regimen of eight-day sustained exposure to IL-12 was developed (1 μg/rat/day), which caused mild side effects, increased baseline resistance to experimental MADB106 metastasis, and abolished the promotion of metastasis by laparotomy and other immunosuppressive paradigms. Depletion of NK cells indicated their major role in controlling MADB106 metastasis in naïve and IL-12 treated rats. Studying NK cytotoxicity, we found that IL-12 did not potentiate activity per NK cell, nor protected it from suppression by surgery. However, IL-12 increased the numbers of NK cells in the circulation and marginating pulmonary pool of naïve and operated rats, and correspondingly increased total NK activity in these compartments. Therefore, this study indicates anti-tumor effects of IL-12 based on increased numbers of strategically located NK cells, and advocates a prophylactic approach against the potential metastasis-promoting effects of surgery.     

血清抗体及补体对利妥昔单抗介导的NK 细胞杀伤Raji细胞作用的影响

 【摘要】 目的 体外检测血清抗体及补体对利妥昔单抗介导的NK细胞对Raji细胞杀伤作用的影响。方法 通过巢式反转录聚合酶链反应（nest-PCR）检测NK细胞上FcγRⅢa（CD16a）基因型;流式细胞术检测加入血清抗体前后NK细胞上FcγRⅢa的表达;DIO-PI双荧光染色法检测添加血清补体及抗体后NK细胞对Raji细胞的杀伤效应强度。结果 添加血清补体组杀伤率较未添加组明显增强,而添加血清抗体组杀伤率较未添加组明显减弱,在FcγRⅢa-158V／V组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（94.25±1.79） %、（59.79±0.66） %和（69.05±2.38） %,三组之间两两比较均P＜0.05;在FcγRⅢa-158V／F组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（66.71±5.57） %、（18.13±2.99） %和（39.63±3.86） %,三组之间两两比较均P＜0.05。结论 血清补体可增强利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。血清抗体会减弱利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。使用肿瘤特异性单克隆抗体（MAb）治疗肿瘤患者时,同时输注新鲜冷冻血浆可提高其抗肿瘤疗效;而MAb与静脉注射用免疫球收白（IVIG）同时使用则可能降低其抗肿瘤疗效。     

Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay

A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.