Semi-automated PCR method for quantitating MDR1 expression

The MDR1 gene is involved in drug resistance in many hematopoietic and solid tumors. The Quantitative PCR System 5000 (QPCR-5000; Perkin-Elmer) is a new instrument system that uses electrochemiluminescence to automatically quantitate polymerase chain reaction (PCR) products. A comparative study between radioactively labeled PCR (32P-PCR) and QPCR was performed to analyze the MDR1 gene expression in the drug-resistant (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 8226/S, CEM Dox1 and three acute myeloid leukemia (AML) patient samples. Using the Dox40 and Dox6 resistant cell lines, we compared the sensitivities of QPCR and 32P-PCR. A strong signal was obtained from QPCR at 20 to 25 cycles (which is in the linear range for quantitation), while a weak signal was obtained using 32P-PCR at the same cycle number. Dilution experiments gave better precision with the QPCR than with the radioactive method. AML samples were studied with the MDR1-specific MAbs MRK16 and 4E3, and the efflux function was analyzed using Rh-123 retention in the absence or presence of verapamil. The three samples showed high (D = 0.79), medium (D = 0.52) and negative (D = 0.08) p-glycoprotein (P-gp) levels and correlated with efflux function. The MDR1/beta 2-M mRNA ratios for 32P-PCR were 0.41, 0.40 and 0.12, respectively, and were 0.127, 0.097 and 0.028, respectively, for QPCR. There were significant differences between the samples with high and medium P-gp levels comparing the two methods. Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR. In conclusion, QPCR was found to be more reproducible, accurate and sensitive than 32P-PCR.     

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A drug-resistant cell line (EAC/Dox) was developed by repeated exposure of Ehrlich ascites carcinoma cells to Doxorubicin (Dox) in vivo in male albino Swiss mice (6-8 weeks old). The weekly i.p. injections of Dox to mice (2 or 4 mg/kg/week for 4 months) gave rise to Dox-resistant cell line EAC/Dox, which displayed typical multidrug resistant (MDR) features of cross-resistance to a number of structurally and functionally unrelated drugs like doxorubicin, vinblastine and cisplatin. Moreover, the EAC/Dox cell line had lower drug accumulation than drug-sensitive (EAC/S) cells. Study of Western blots and immunofluorescence revealed that P-glycoprotein 170 kDa (P-gp) was absent in EAC/Dox cells. The drug resistance appeared to be due to the presence of a higher level of reduced glutathione (GSH) and glutathione S-transferase (GST) in EAC/Dox cells than in drug-sensitive (EAC/S) cells. The two structurally similar hydroxamic acid derivatives, i.e. oxalyl bis(N-phenyl)hydroxamic acid (X1) and succinyl bis(N-phenyl)hydroxamic acid (X2), having very low in vitro toxicity (IC50 value 250 microg/ ml), were investigated for their efficacy to reverse MDR. The compound X1 was able to reverse the effect of MDR and reduce GST in EAC/Dox cells. The compound X2 had no ability to reverse the effect of MDR. Further study on the mechanism of glutathione depletion and the resistance modifying property of X1 on other cell lines is warranted.     

Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.     

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.     

In vitro effect of GF120918, a novel reversal agent of multidrug resistance, on acute leukemia and multiple myeloma cells

Resistance to chemotherapy in multiple myeloma (MM) and acute myeloid leukemia (AML) is frequently caused by multiple drug resistance (MDR), characterized by a decreased intracellular drug accumulation. MDR is associated with expression of P-glycoprotein (P-gp). GF120918, an acridine derivative, enhances doxorubicin cell kill in resistant cell lines. In this study, the effect of GF120918 on MDR cell lines and fresh human leukemia and myeloma cells was investigated. The reduced net intracellular rhodamine-123 (Rh-123) accumulation in the MDR cell lines RPMI 8226/Dox1, /Dox4, /Dox6 and /Dox40 as compared with wild-type 8226/S was reversed by GF120918 (0.5-1.0 microM), and complete inhibition of rhodamine efflux was achieved at 1-2 microM. This effect could be maintained in drug-free medium for at least 5 h. GF120918 reversal activity was significantly reduced with a maximum of 70% in cells incubated with up to 100% serum. GF120918 significantly augmented Rh-123 accumulation in vitro in CD34-positive acute leukemia (AML) blasts and CD38-positive myeloma (MM) plasma cells obtained from 11/27 de novo AML and 2/12 refractory MM patients. A significant correlation was observed between a high P-gp expression and GF120918 induced Rh-123 reversal (P=0.0001). Using a MRK16/IgG2a ratio > or = 1.1, samples could be identified with a high probability of GF120918 reversal of Rh-123 accumulation. In conclusion, GF120918 is a promising MDR reversal agent which is active at clinically achievable serum concentrations.     

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.     

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The sensitivity of 18 permanent hemopoietic cell lines to Cyclosporin A (CsA) was tested in a 3H-thymidine incorporation rate assay. Two human T cell lines (Molt4 and CEM) were significantly inhibited by a CsA concentration of 0.5 microgram/ml. Not affected at all or only inhibited by 10 to 20 times higher CsA concentrations were: three human B cell lines (4413a, Daudi, Raji), a monkey B cell line (B95-8), a mouse plasmocytoma line (X63-Ag8/653), a human non-B T cell line (Reh), four human myeloid lines (HL-60, ML-1, ML-2, ML-3), a human myelomonocytic line (Karpas 230), four human monoblastic lines (U 937, SU-DHL-1, THP-1, Karpas 241) and a human erythroid line (K 562). It therefore seems that among permanently growing hemopoietic cells a cell type specificity for T cells also exists.     

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.     

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The comet test is a reported method for measuring DNA damage in individual mammalian cells. In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated. For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents. To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR. Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested. Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER). Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested. Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.     

Sensitivity of vincristine-sensitive K562 and vincristine-resistant K562-Lucena 1 cells to anthracyclines and reversal of multidrug resistance

The phenomenon of multidrug resistance (MDR), that involves the efflux pump P-glycoprotein, can be reversed by a number of substances known as MDR modulators or reversing agents. In the present study we investigated the action of three anthracyclines, mitoxantrone and vincristine on short-term (72 h) cultures using 2 methods ([3H] incorporation and MTT (3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrazolium bromide)), on 2 cell lines: K562, a human erythroleukemia, and a vincristine-resistant subline K562-Lucena 1. Using the same culture methods plus flow cytometry analysis, the reversing potentials of cyclosporin A and verapamil were studied in both cell lines. There were differences in the sensitivity and resistance profiles of the two lines to the various drugs but daunorubicin (5 micrograms/ml) and idarubicin (0.035 micrograms/ml) were the most effective when each was used in high concentration. Cyclosporine at 200 ng/ml and verapamil at 5 micrograms/ml reversed MDR in the resistant line, and had a synergistic action with chemotherapeutic agents on the sensitive line. Again differences were demonstrable between combinations of the various drugs and reversal was only clearly shown with the method measuring cell proliferation ([3H] incorporation) but not by the method measuring metabolic activity (MTT). The efflux of rhodamine-123 mimics the functional activity of the pump and cyclosporine was a better reversing agent by this criteria. These data show that the results obtained in in vitro studies attempting to identify treatments for different types of leukemias depend to a large extent on the methods used to measure cell response.     

Antitumor activity of free and liposome-entrapped annamycin, a lipophilic anthracycline antibiotic with non-cross-resistance properties

The lipophilic anthracycline antibiotic annamycin (Ann) was entrapped in liposomes of different size [median diameter: 1.64 microns, multilamellar liposomal Ann (L-Ann); 0.030 micron, small unilamellar Ann (S-Ann)] with > 90% entrapment efficiency and tested in vitro against four pairs of sensitive and multidrug-resistant (MDR) tumor cell lines and in vivo by the i.v. route in five tumor models: advanced s.c. B16 melanoma; s.c. M5076 reticulosarcoma; lung metastases of Lewis lung carcinoma; and s.c. KB and KB-V1 xenografts in nude mice. Predetermined optimal doses of the different formulations were used and the results were compared with doxorubicin (Dox). In vitro, Ann, either in suspension in 10% dimethyl sulfoxide (F-Ann) (1 mg/ml) or entrapped in liposomes, was able to partially overcome resistance in all four pairs of sensitive and MDR KB, 8226, P388, and CEM cell lines (resistance indexes 63, 269, 333, and 356 for Dox versus 4, 5, 19, and 8.7 for L-Ann, respectively). In vivo, both F-Ann and liposome-entrapped Ann were slightly more effective than Dox in inhibiting the growth of advanced s.c. B16 melanoma tumors. L-Ann was markedly more effective than Dox and moderately more effective than F-Ann in prolonging the life span of animals bearing s.c. M5076 and lung metastases of Lewis lung carcinoma tumors. All drugs were equally effective at optimal doses in delaying the growth of s.c. KB xenografts, whereas all Ann formulations were markedly more effective than Dox in delaying the growth of s.c. KB-V1 (MDR) xenografts. In all in vivo experiments, S-Ann was consistently more effective than L-Ann and L-Ann was more effective than F-Ann. These results indicate that (a) Ann is more effective than Dox by the i.v. route against several tumor models and that MDR tumors are partially not cross-resistant to Ann both in vitro and in vivo, (b) liposomes enhance the in vivo antitumor properties of Ann, and (c) small liposomes are more effective than large liposomes in enhancing Ann antitumor activity.     

Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.     

The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy-D-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.     

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OBJECTIVE: Water is widely used as a tumoricidal agent during transurethral resection of bladder tumour (TURBT). Despite this, recurrences are common. One possible explanation may be that while the parental bladder cancer cells are sensitive to water, their multidrug resistant (MDR) clones which occur commonly, may be resistant to it. If so, is this a preserve of the MDR phenotype or do other mechanisms confer resistance to water? METHODS: The parental human urothelial cancer cell lines (MGHU-1 and RT112) and their drug-resistant variants (MDR and non-MDR) were exposed to short pulses of water. The MDR status of these cells was verified using confocal microscopy to detect intracellular accumulation of fluorescent drug (epirubicin). A clonogenic assay was used to establish tumoricidal efficacy on cells in suspension. A thiozolyl blue (MTT) assay was used on adherent cells to assess the residual viable biomass. RESULTS: In the MDR clone of cells, colony counts were reduced by 20% after treatment with water as compared to a 60% reduction in the parental cell line. There was a similar reduction in colony counts between the parental RT112 and its non-MDR cisplatin resistant variant. CONCLUSION: Water is less effective on MDR cells. Resistance to water may be a function of the MDR phenotype.     

Refined localization of the asparagine synthetase gene (ASNS) to chromosome 7, region q21.3, and characterization of the somatic cell hybrid line 4AF/106/KO15

We have mapped the asparagine synthetase gene (ASNS) to 7q21.3 by fluorescence in situ hybridization. While this study refined the localization of the gene, it also revealed a rearrangement in a somatic cell hybrid line which was used in previous ASNS mapping. Using additional probes from other regions of human chromosome 7, we showed that this cell line (4AF/106/KO15) contained a rearranged chromosome 7 in which a segment of the long arm was apparently duplicated and inserted into the short arm. Caution should be used therefore when interpreting data obtained from this cell line for gene mapping studies.     

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We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line). Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold). Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line. This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multidrug-resistance mechanism. Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min. This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 0.5 cells. Northern and Western blot analysis of the mdrI gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdrI gene and the P-glycoprotein to a slightly higher level than the C6 IV line. Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdrIa gene, whereas the C6 IV cells preferentially over-expressed the mdrIb gene. This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines. It can be concluded therefore that the mdrIa gene product is more efficient than the mdrIb gene product in extruding anti-cancer drugs from the cells; and that the mdrIb gene product might preferentially extrude vincristine rather than doxorubicin.     

The N-7-substituted acyclic nucleoside analog 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine is a potent and selective inhibitor of herpesvirus replication

2-Amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) represents the first antivirally active nucleoside analog with the side chain attached to the N-7 position of the purine ring. Compound S2242 strongly inhibits the in vitro replication of both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (50% effective concentration [EC50], 0.1 to 0.2 microgram/ml), varicella-zoster virus (EC50, 0.01 to 0.02 microgram/ml) and thymidine kinase (TK)-deficient strains of HSV (EC50, 0.4 microgram/ml) and varicella-zoster virus (EC50, 0.2 to 0.5 microgram/ml). Potent activity was also observed against murine cytomegalovirus (EC50, 1 microgram/ml), human cytomegalovirus (HCMV) (EC50, 0.04 to 0.1 microgram/ml), and human herpesvirus 6 (EC50, 0.0005 microgram/ml). Compound S2242 (i) was not cytotoxic to confluent Vero, HeLa, or human fibroblast cells at concentrations of > 100 micrograms/ml, (ii) proved somewhat more cytostatic to Vero, HEL, HeLa, and C127I cells than ganciclovir, and (iii) was markedly more cytostatic than ganciclovir to the growth of the human lymphocytic cell lines HSB-2 and CEM degrees. In contrast to ganciclovir, (i) compound S2242 proved not to be cytocidal to murine mammary carcinoma (FM3A) cells transfected with the HSV-1 or HSV-2 TK gene, (ii) exogenously added thymidine had only a limited effect on its anti-HSV-1 activity, and (iii) the compound was not phosphorylated by HSV-1-encoded TK derived from HSV-1 TK-transfected FM3A cells, indicating that the compound is not activated by a virally encoded TK. Compound S2242 inhibited (i) the expression of late HCHV antigens at an EC50 of 0.07 microgram/ml (0.6 microgram/ml for ganciclovir) and (ii) HCMV DNA synthesis at an EC50 of 0.1 microgram/ml (0.32 microgram/ml for ganciclovir), i.e., values that are close to the EC50S for inhibition of HCMV-induced cytopathogenicity. Neither ganciclovir nor S2242 had any effect on the expression of immediate-early HCMV antigens, which occurs before viral DNA synthesis. In time-of-addition experiments, S2242 behaved like ganciclovir and acyclovir; i.e., the addition of the drugs could be delayed until the onset of viral DNA synthesis.