International round-robin experiment for angle-resolved light scattering measurement.

An international round-robin experiment has been conducted to test procedures and methods for the measurement of angle-resolved light scattering. ASTM E2387-05 has been used as the main guide, while the experience gained should also contribute to the new ISO standard of angle-resolved scattering currently under development (ISO/WD 19986:2016). Seven laboratories from Europe and the United States measured the angle-resolved scattering from Al/SiO2-coated substrates, transparent substrates, volume diffusors, quasi-volume diffusors, white calibration standards, and grating samples at laser wavelengths in the UV, VIS, and NIR spectra. Results were sent to Fraunhofer IOF, which coordinated the experiments and analyzed the data, while ESA-ESTEC, as the project donor, defined conditions and parameters. Depending mainly on the sample type, overall good to reasonable agreements were observed, with largest deviations at scattering angles very close to the specular beam. Volume diffusor characterization unexpectedly turned out to be challenging. Not all participants provided measurement uncertainty ranges according to the Guide to the Expression of Uncertainty in Measurement; often, a single general scatterometer-related measurement uncertainty value was stated. Although relative instrument measurement uncertainties close to 1% are sometimes claimed, the comparison results did not support these claims for specular scattering samples as mirrors, substrates, or gratings.

Efficient syntheses of (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine; a nucleoside analogue with potent biological activity.

(E)-5-(2-Bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) is a potent antiherpesvirus agent and its use in gene therapy as an anticancer agent has recently been described. We here outline 2 efficient methods for the synthesis of S-BVDU. The decision as to which method is to be used depends upon the starting materials available but starting from BVU, an overall yield of beta-nucleoside of 35% can be expected. From 5-ethyl-2'-deoxy-4'-thiouridine, radical bromination using bromine will give a quantitative conversion to S-BVDU if unreacted starting material is recycled (50%) or using N-bromosuccinimide, a one step yield in excess of 80% can be obtained.

Analogues of 10-deazaaminopterin and 5-alkyl-5,10-dideazaaminopterin with the 4-substituted 1-naphthoyl group in the place of 4-substituted benzoyl.

10-Deaza modifications of classical antifolate analogues bearing the 1,4-disubstituted naphthalene ring in place of the 1,4-disubstituted benzene ring were prepared and tested for antitumor activity. Naphthalene analogues (9a-c, respectively) of 10-deazaaminopterin, 5-methyl-5, 10-dideazaaminopterin, and 5-ethyl-5,10-dideazaaminopterin were prepared by a route consisting of C-alkylations of the anion derived from 4-carboxyl-1-naphthaleneacetic acid dimethyl ester (2) by 6-(bromomethyl)-2,4-diaminopteridine (1a) and 6-(bromomethyl)-2,4-diamino-5-methyl- and -5-deazapteridines (1b and 1c, respectively) followed by ester hydrolysis and subsequent decarboxylation to give naphthalene analogues (7a-c, respectively) of 4-amino-4-deoxy-10-deazapteroic acid and 4-amino-4-deoxy-5- methyl- and -5-ethyl-5,10-dideazapteroic acids. Peptide coupling of 7a-c with L-glutamic acid dialkyl ester followed by mild ester hydrolysis gave target compounds 9a-c. The key advantage of this route is circumvention of a hydrogenation step requiring selectivity as in earlier approaches involving 9,10-olefinic precursors. Steric limitations thwarted plans to prepare the naphthalene analogue of 10-ethyl-10-deazaaminopterin; attempted alkylations of 2-(4-carboxy-1-naphthyl)butyric acid dimethyl ester with 1a failed as did attempted further alkylation (by EtBr) of the product derived from 1a and 2. Growth inhibition tests against three tumor cell lines (L1210, S180, and HL60) showed 9a to be 4-6-fold more inhibitory than methotrexate but not as inhibitory as 10-ethyl-10-deazaaminopterin; 9b and 9c were no more inhibitory than MTX. In tests against the EO771 mammary adenocarcinoma in mice, 9a was less active than MTX.

Schedule-dependent synergism of taxol or taxotere with edatrexate against human breast cancer cells in vitro.

A new dihydrofolate reductase inhibitor, edatrexate (EDX), and the microtubule polymerization promotor, taxol (TXL) or taxotere (TXT), each have significant therapeutic activity against human breast cancer in clinical trials. Since they also have distinctly different mechanisms of actions and have mainly non-overlapping toxicities, they may be effective in combination in the treatment of this disorder. Schedule-dependent interactions between these taxanes and EDX against human breast adenocarcinoma cells (SK-Br-3) were quantitatively assessed in vitro to determine whether these interactions are synergistic or antagonistic. SK-Br-3 cells were grown as a monolayer in 96-well microplates. The dose-effect relationships of the drugs, singly and in combination, in inhibiting the growth over a 7-day period were determined by the SRB protein staining assays. Cell cultures were exposed to drug as a 3-h pulse at either 0-3 h or 24-27 h. Synergism or antagonism at different concentrations and at different effect levels were assessed with the median effect principle and the combination index-isobologram method using computer software. These methods were selected because they take into account both the potencies and the shape of the dose-effect curves. Exposure of cells to an equimolar combination of EDX + TXL (0-3 h) resulted in synergism at high effect levels. Pretreatment of cells with EDX (0-3 h) followed by TXL (24-27 h) showed even greater synergism in inhibiting cell growth. Moderate antagonism was observed with the reverse schedule. EDX + TXT (0-3 h) was additive, but pretreatment with EDX (0-3 hr) followed by TXT (24-27 h) showed synergism. However, the reverse order showed antagonism. Studies on another breast tumor cell line, ZR-57-1, also showed the schedule of EDX (0-3 h) + TXT or TXL (24-27 h) to be more synergistic than, the other two schedules examined. These results show potent schedule-dependent synergism of the combinations of TXL or TXT with EDX, and should form a rationale for designing clinical protocols utilizing these agents particularly for the treatment of breast cancer patients.

9-substituted acridine derivatives with long half-life and potent antitumor activity: synthesis and structure-activity relationships.

A series of DNA-intercalating 9-anilinoacridines, namely 9-phenoxyacridines, 9-(phenylthio)acridines, and 9-(3',5'-disubstituted anilino)acridines, were synthesized as potential antitumor agents with inhibitory effects on DNA topoisomerase II. Unlike amsacrine (m-AMSA), these agents were designed to avoid the oxidative metabolic pathway. These acridine derivatives were, therefore, expected to have long half-life in plasma. Both 9-phenoxyacridines and 9-(phenylthio)acridines were found to have moderate cytotoxicity against mouse leukemia L1210 and human leukemic HL-60 cell growth in culture. Among 9-(3',5'-disubstituted anilino)acridines, 3-(9-acridinylamino)-5-(hydroxymethyl)aniline (AHMA) was found to be a potent topoisomerase II inhibitor and exhibited significant antitumor efficacy both in vitro and in vivo. Chemotherapy of solid-tumor-bearing mice with 10, 10, and 5 mg/kg (QD x 4, ip) AHMA, VP-16, and m-AMSA, respectively, resulted in more tumor volume reduction by AHMA than by VP-16 or m-AMSA for E0771 mammary adenocarcinoma and B-16 melanoma. For Lewis lung carcinoma, AHMA was as potent as VP-16 but more active than m-AMSA. Structure-activity relationships of AHMA derivatives are discussed.

Effective combination therapy of metastatic murine solid tumors with edatrexate and the vinca alkaloids, vinblastine, navelbine and vindesine.

Studies are described in which a new folate analogue, edatrexate (EDX), in combination with the vinca alkaloids, vinblastine (VBL), navelbine (NVB) or vindesine (DVA) was evaluated against E0771 mammary adenocarcinoma, T241 fibrosarcoma and the Lewis lung tumor. Each of the four agents when given individually to animals 3 days after transplant of these tumors resulted in increases in survival of 53-143%. The relative effectiveness of these agents was (in increasing order) VBL, NVB congruent to DVA, EDX, with no long-term survivors obtained with any. Combination therapy with EDX and vinca alkaloids required dosage attenuation but was still markedly more effective. Treatment of E0771 and T241 tumors with EDX and either NVB or DVA increased survival 3- to 4-fold compared with therapy with individual agents and yielded 40-70% long-term survivors, while EDX with VBL increased survival 2- to 3-fold and yielded 20-40% long-term survivors. Simultaneous or sequential (EDX given 24 h before vinca alkaloid) administration of combined therapy was equally effective. Sequential administration of these agents at the same doses in the reverse order was highly toxic and required further dosage attenuation which compromised efficacy. Effects of these combinations against the Lewis Lung tumor were not as pronounced and were somewhat schedule-dependent. Simultaneous administration of EDX with VBL, NVB or DVA increased survival 2- to 3-fold over that obtained with single agents alone and yielded 10-40% long-term survivors, while sequential administration increased survival < 2-fold over that obtained with single agents and yielded 0-20% long-term survivors. These results suggest that combined therapy with these agents in patients may have appreciable utility and provide a basis for further clinical trials.

Disposition of leucovorin and its metabolites in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of methotrexate-pretreated mice.

Leucovorin (LV or 5-CHOFH4) has had long-standing clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH2FH4) plus tetrahydrofolate (FH4) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH4). The dihydrofolate (FH2) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH3FH4) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH4) in both intestinal epithelial and L1210 cells. The elevation of FH2 in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH2FH4 + FH4. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.

Studies on analogues of classical antifolates bearing the naphthoyl group in place of benzoyl in the side chain.

Analogues of classical antifolates with the 4-aminobenzoyl group replaced by 4-amino-1-naphthoyl were synthesized for study after molecular modeling indicated ample spatial accommodation for the naphthalene ring and even larger groups in models based on reported X-ray crystallographic data describing the binding of methotrexate to human dihydrofolate reductase (DHFR). The side-chain precursors, N-(4-amino- and 4-(methylamino)-1-naphthoyl)-L-glutamic acid diethyl esters, were synthesized, and the 2,4-diamino-substituted heterocyclic groups were attached using several methods. Target compounds included naphthoyl analogues of aminopterin (AMT), methotrexate (MTX), 5-deazaAMT, 5-deazaMTX, 5-methyl-5-deazaAMT, 5-methyl-5-deazaMTX, and 5,8-dideazaAMT. A 5,6,7,8-tetrahydronaphthoyl analogue of 5-deazaAMT was also prepared. None of the naphthoyl analogues showed loss in binding to DHFR compared with the corresponding antifolate bearing the benzoyl group, thus confirming the anticipated bulk tolerance. Only the 5,6,7,8-tetrahydronaphthoyl analogue displayed reduced antifolate effects. Substrate activity toward folylpolyglutamate synthetase was, however, severely compromised. The naphthoyl compounds were transported into L1210 cells 3-6 times more readily than MTX, and despite apparently low levels of intracellular polyglutamylation, each compound was found to be significantly more potent than MTX in inhibiting tumor cell growth in vitro in three lines (L1210, HL60, and S180). The MTX, 5-methyl-5-deazaAMT, and 5-methyl-5-deazaMTX analogues were evaluated in vivo alongside MTX against E0771 mammary adenocarcinoma in mice. All three proved more effective than MTX in retarding the tumor growth. The naphthoyl analogue of 5-deazaAMT strongly inhibited DHFR from Pneumocystis carinii, Toxoplasma gondii, and rat liver giving IC50 (pM) values of 0.53, 2.1, and 1.6 respectively, but this compound did not inhibit in vitro growth of T. gondii, thus indicating lack of transport.

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.

Markedly improved efficacy of edatrexate compared to methotrexate in a high-dose regimen with leucovorin rescue against metastatic murine solid tumors.

10-Ethyl-10-deazaaminopterin (EDX, edatrexate) exhibits therapeutic activity against methotrexate (MTX)-resistant tumors in animals and patients. In an effort to improve its efficacy among more chemoresistant tumors, studies were initiated in murine models of advanced metastatic disease comparing EDX and MTX at their maximum tolerated dose alone and in a high-dose regimen incorporating low-dose, delayed Ca leucovorin (LCV) rescue. Both twice-weekly x 3 and weekly x 3 schedules of administration were used with LCV given 16, 20, and 24 h after EDX. The LCV dose required to protect mice was 1/40 and 1/20 of the EDX or MTX dose, respectively, on either schedule. Therapy was initiated 5 or 6 days following i.v. implant of 5 x 10(5) cells of the E0771 mammary adenocarcinoma, T241 fibrosarcoma, Lewis lung carcinoma, B16 melanoma, or C38 colon carcinoma. MTX was essentially ineffective (increase in life span = < 30%) when given alone and either ineffective or only modestly effective (increase in life span = 20-80%) in increasing survival when given in the high-dose regimen to tumor-bearing mice. EDX alone was more effective than MTX when it was given in either regimen of therapy. Also, EDX given in the high-dose regimen (either twice-weekly or weekly x 3) was markedly more effective than EDX alone. Increased survival with this regimen was 2-3-fold greater than EDX alone against all 5 tumors, and long-term survivors were obtained with E0771 (20%), T241 (30-40%), Lewis lung (10-15%), B16 (20%), and C38 (40%) tumors. The administration of 6 doses rather than 3 doses on the twice-weekly schedule against T241 and Lewis lung tumors required a modest increase in the LCV dose but substantially improved efficacy, with as much as 70% long-term survivors (T241 tumor). We conclude that the use of a high-dose regimen with delayed LCV rescue markedly improved the therapeutic effectiveness of EDX against advanced metastatic disease in tumor-bearing mice. These studies should provide a framework for further clinical work with EDX, using this modality of therapy.

Synthesis and antifolate evaluation of 10-ethyl-5-methyl-5,10- dideazaaminopterin and an alternative synthesis of 10-ethyl-10- deazaaminopterin (edatrexate).

Previous findings suggesting that 5,10-dialkyl-substituted derivatives of 5,10-dideazaaminopterin warranted study as potential antifolates prompted synthesis of 10-ethyl-5-methyl-5,10- dideazaaminopterin (12a). The key step in the synthetic route to 12a was Wittig condensation of the tributylphosphorane derived from 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (7a) with methyl 4-propionylbenzoate. Reaction conditions for the Wittig condensation were developed using the tributylphosphorane prepared from 6-(bromomethyl)-2,4-pteridinediamine (7b) as a model. Each of the respective Wittig products 8a and 8b was obtained in 75-80% yield. Hydrogenation of 8a and 8b at their 9,10-double bond afforded 4-amino-4-deoxy-10-ethyl-5-methyl-5,10-dideazapteroic acid methyl ester (9a) and 4-amino-4-deoxy-10-ethyl-10-deazapteroic acid methyl ester (9b). This route to 9b intersects reported synthetic approaches leading to 10-ethyl-10-deazaaminopterin (10-EDAM, edatrexate), an agent now in advanced clinical trials. Thus the Wittig approach affords an alternative synthetic route to 10-EDAM. Remaining steps were ester hydrolysis of 9a,b to give carboxylic acids 10a,b followed by standard peptide coupling with diethyl L-glutamate to produce diethyl esters 11a,b, which on hydrolysis gave 12a and 10-EDAM (12b), respectively. The relative influx of 12a was enhanced about 3.2-fold over MTX, but as an inhibitor of dihydrofolate reductase (DHFR) from L1210 cells and in the inhibition of L1210 cell growth in vitro, this compound was approximately 20-fold less effective than MTX (DHFR inhibition, Ki = 4.82 +/- 0.60 pM for MTX, 100 pM for 12a; cell growth, IC50 = 3.4 +/- 1.0 nM for MTX, 65 +/- 18 nM for 12a).

Preferential selection during therapy in vivo by edatrexate compared to methotrexate of resistant L1210 cell variants with decreased folylpolyglutamate synthetase activity.

Acquired resistance of the L1210 leukemia in mice developed with less rapidity during therapy with edatrexate (10-ethyl-10-deazaaminopterin, EDX) than with MTX. Since this was explained only partially by the somewhat greater antitumor activity of EDX, this result may also reflect a difference in biochemical phenotypes selected in each case. Among 20 sublines selected for resistance to MTX, a reduction in influx, an elevation of DHFR, and a reduction of DHFR inhibition by MTX were all delineated. Among 14 sublines selected for resistance to EDX, both a reduction in influx and an elevation in level of DHFR were also commonly found. In addition, however, 7 of 14 EDX-resistant sublines exhibited a reduction in the level of folylpolyglutamate synthetase (FPGS) activity. Clonal derivatives of these 7 EDX-resistant cell lines exhibited 2- to 28-fold reductions in FPGS activity and a commensurate reduction in [3H]-MTX polyglutamate formation in situ following exposure to [3H]-MTX during growth in mice. An analysis of the kinetics and relative substrate preferences for FPGS from variant and parental L1210 cells revealed that the various changes in FPGS activity were at the level of the Vmax rather than Km. These results derived from an in vivo tumor model provide further evidence for a role of FPGS as a determinant of cytotoxicity and acquired resistance to classical folate analogs. They also provide evidence in the same pharmacologic model for a manifestation of resistance to 4-aminofolates in vivo that involves all of the alterations of its primary target, transport, and metabolism that have ever been associated with acquired resistance in cell culture systems.

Structural design, biochemical properties, and evidence for improved therapeutic activity of 5-alkyl derivatives of 5-deazaaminopterin and 5-deazamethotrexate compared to methotrexate in murine tumor models.

Studies are described examining a new class of 4-aminofolate analogues modified by an N to C conversion and alkyl substitution at the N-5 position of aminopterin and methotrexate. All of these analogues were equivalent to aminopterin and methotrexate as inhibitors of tumor cell dihydrofolate reductase (Ki = 3.49-5.16 pM). N to C conversion at the N-5 position of aminopterin reduced its influx (inferred from the change in Ki) 3-fold, but the same modification increased influx of methotrexate 2-3-fold in Sarcoma 180 cells. Alkylation (methyl or ethyl) of this position on 5-deazaaminopterin increased influx 3-fold, while a similar alteration of 5-deazamethotrexate increased influx 4-5-fold. Influx of the methotrexate analogues was increased a total of 14-fold as a result of these modifications. Similar differences among these analogues were observed for inhibition of Sarcoma 180 cell growth in culture. Inhibitory potency was in the ascending order methotrexate less than 5-deazamethotrexate less than 5-deazaaminopterin less than aminopterin less than 5-alkyl (methyl or ethyl) analogues of 5-deazaaminopterin and 5-deazamethotrexate (the ethyl analogues were 2-fold more inhibitory than the methyl analogues). All of the analogues examined were equivalent in regard to efflux from Sarcoma 180 cells. Differences in transport alone did not account for all of the increased inhibitory potency (up to 33-fold) of the 5-alkyl-5-deaza analogues compared to the parent compounds. The extent of polyglutamylation of 5-deazaaminopterin and 5-deazamethotrexate and their 5-alkyl derivatives in Sarcoma 180 cells was substantially less compared to aminopterin and equivalent to methotrexate. Transport inward of 5-deazaaminopterin in isolated crypt cell epithelium from mouse small intestine was 2-fold lower than aminopterin (influx Km = 14.2 +/- 2 microM), while influx of 5-deazamethotrexate was 2-fold greater than methotrexate (influx Km = 98.6 +/- 23). However, transport inward of all of the 5-alkyl derivatives of these 5-deaza analogues was intermediate [influx Km = 44.4 +/- 11 (SEM) to 49.8 +/- 12 microM] between values for aminopterin and methotrexate. These differences accounted, to some extent, for the reduced toxicity of the 5-alkyl-5-deazaaminopterin analogues compared to aminopterin and the increased toxicity of 5-methyl-5-deazamethotrexate compared to methotrexate. All of the 5-alkyl derivatives of aminopterin and methotrexate were more active in vivo than methotrexate against four murine tumor models.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical synthesis and biological activities of 5-deazaaminopterin analogues bearing substituent(s) at the 5- and/or 7-position(s).

Condensation of cyanothioacetamide (4) with ethyl alpha-(ethoxymethylene)acetoacetate (5b), ethyl 4-ethoxy-2-(ethoxymethylene)-3-oxobutanoate (5c), ethyl 2-(ethoxymethylene)-3-oxo-4-phenylpropanoate (5d) afforded exclusively the corresponding 6-substituted pyridines (6b-d). Cyclization of 4 with 3-carbethoxybutane-2,4-dione (5e) gave 3-cyano-5-(ethoxycarbonyl)-4,6-dimethylpyridine-2(1H)-thione (6e), whereas reaction of 4 with 3-carbethoxy-1-phenylpropane-1,3-dione (5f) yielded two products, 3-cyano-5-(ethoxycarbonyl)-4-methyl-6-phenylpyridine-2(1H)-thione (6f) and the 6-methyl-4-phenyl isomer 6g. The structural assignments for 6f and 6g are made on the basis of 1H and 13C NMR spectral analyses of the 2-(methylthio)nicotinates (7f,g) prepared from 6f and 6g by treatment with MeI/K2CO3. Nicotinates 7b,d-g were converted into their corresponding 2,4-diaminopyrido[2,3-d]pyrimidines 12b,d-g in five steps, via reduction, protection, oxidation, condensation with guanidine, and deprotection. The 7-mono- and 5,7-disubstituted-5-deazaaminopterins (1b,d-g) were prepared from the respective pyrido[2,3-d]pyrimidines 12b,d-g. Preliminary biological studies showed that 7-methyl and 5,7-dimethyl analogues (1b and 1e) were less active than methotrexate against human leukemic HL-60 and murine L-1210 cells in tissue culture. Compound 1e produced an ILS of 71% at 100 mg/kg per day X 5 (ip) in BDF mice inoculated ip with 10(6) L-1210 cells.

L1210 leukemia hybrids isolated after fusion of alkylating agent-resistant sublines.

The two resistant lines, L1210/CPA (cyclophosphamide) and L1210/MeCCNU (1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea) were used, each of which is not cross-resistant to the drug to which the other line is resistant. Their resistance was used as markers as well as the basis for selection of the hybrids. For the production of hybrids five in-vivo or in-vitro schedules were employed. The in-vitro methods produced six successful hybrid lines, but the in-vivo schedules produced none. Resistance to both CPA and MeCCNU was expressed dominantly in the hybrids. The hybrids had chromosome modes ranging from 68 to 78. This study shows that CPA and MeCCNU can be used both as markers and as selective agents, and that CPA and MeCCNU resistance in L1210 leukemia are dominantly expressed in the hybrid.