Transport of nitrogen mustard on the transport-carrier for choline in L5178Y lymphoblasts.

The uptake of choline by L5178Y lymphoblasts occurs by a carrier mechanism and is an active process. Transport of nitrogen mustard and its hydrolyzed derivative is mediated by this same carrier. This finding is an example of drug transport by a carrier whose primary function is transport of a naturally occurring substrate.

Glutathione S-transferase activity, sulfhydryl group and glutathione levels, and DNA cross-linking activity with chlorambucil in chronic lymphocytic leukemia.

Glutathione (GSH) levels and glutathione S-transferase (GST) activities were measured in the leukemia cells of 12 patients with chronic lymphocytic leukemia. Both were correlated with prior clinical exposure to alkylating agents and with DNA cross-link formation by chlorambucil in these cells in vitro. No correlation was observed between prior exposure to alkylating agents and GSH level or GST activity. An inverse correlation was observed between GST activity and cross-linking by chlorambucil, which was enhanced if both GST activity and GSH level were related to cross-linking. These findings suggest that the combination of GST and GSH protects the DNA of leukemia cells from chlorambucil, but the role of this combination in clinical resistance remains to be determined.

The role of drug transport in resistance to nitrogen mustard and other alkylating agents in L518Y lymphoblsts.

An investigation was undertaken of the mechanism of resistance to nitrogen mustard (HN2) and other alkylating agents, with particular emphasis on the interaction between cross-resistance and drug transport mechanisms in L5178Y lymphoblasts. Dose-survival curves demonstrated that the D0 for HN2-sensitive cells (L5178Y) treated with HN2 in vitro was 9.79 ng/ml and the D0 for HN2-resistant cells (L5178Y/HN2) was 181.11 ng/ml; thus, sensitive cells were 18.5-fold more responsive than were resistant cells and the difference was highly significant (p less than 0.001). A similar evaluation of 5 additional alkylating agents, including chlorambucil, melphalan, 1,3-bis(2-chloroethyl)-l-nitrosourea, Mitomycin C, and 2,3,5-tris(ethyleneimino)-1,4-benzoquinone, revealed that L5178Y/HN2 cells were also cross-resistant, in part, to each of these compounds. Furthermore, the degree of cross-resistance was remarkably similar; for each drug, dose-survival studies showed that HN2-resistant cells were approximately 2- to 3-fold more resistant to therapy than were sensitive cells. L5178Y/HN2 cells were also cross-resistant to cyclophosphamide in vivo; after treatment with cyclophosphamide, DBA/2 female mice that were given inoculations of L5178Y cells, but not those given transplants of L5178Y/HN2 cells, showed a significant prolongation of survival time (p less than 0.01). Transport of HN2, hydrolyzed derivative of HN2 and choline by L5178Y lymphoblasts in vitro was not competitively inhibited by any of the other alkylating agents, suggesting that transport of these compounds was by an independent mechanism. These findings suggest that the mechanism whereby L5178Y/HN2 cells are cross-resistant to other alkylating agents may involve nontransport factors and that these other drugs may bypass a major portion of HN2 resistance by using independent transport systems.

Drug-induced stimulation of transport of hydrolyzed nitrogen mustard and choline by normal and leukemic human cells in vitro.

The effect of morphine and cocaine on the transport of hydrolyzed nitrogen mustard (NH2-OH) and choline by peripheral blood cells of normal subjects and patients with chronic lymphocytic leukemia, acute lymphoblastic leukemia, and acute myeloblastic leukemia was determined. Transport of HN2-OH by lymphocytes from normal individuals and patients with chronic lymphocytic leukemia was stimulated by morphine and cocaine and, in each case, the effect was statistically significant (P less than 0.05 or greater). However, choline transport by normal lymphocytes was not altered by cocaine and was only slightly stimulated by morphine; choline transport by lymphocytes from patients with chronic lymphocytic leukemia was not stimulated by either morphine or cocaine. HN2-OH and choline transport by cells from patients with either acute lymphoblastic or myeloblastic leukemia was stimulated to a comparable degree by both drugs. Stimulation of HN2-OH transport by morphine and cocaine was greater in normal lymphocytes than in acute leukemic cells and the differences were highly significant (p less than 0.001). Conversely, stimulation of choline transport was more marked in acute leukemic cells than in normal lymphocytes, and these differences were also highly significant (p less than 0.001). It was previously shown that transport of nitrogen mustard by normal and leukemic human cells was biphasic in nature, consisting of a choline-independent component at "high" drug concentrations and a choline-dependent system at "low" substrate concentrations. The preferential stimulation of the low-dose, choline-dependent system by morphine and cocaine in acute leukemic cells relative to that observed in normal lymphocytes suggests a possible mechanism of increasing the therapeutic index of nitrogen mustard.

Peripheral leukocyte migration inhibition reactivity to breast cancer antigens in patients with breast cancer and in normal controls.

The peripheral leukocyte migration inhibition test has been used to assess cellular immunity to soluble antigen extracts of breast cancer in patients and normal controls. In sequential tests over several weeks, 23 of 23 patients with breast cancer in remission reacted intermittently, with 67 of 139 tests (48%) being positive (greater than or equal to 20% migration inhibition). Similarly, 6 of 10 patients in relapse reacted intermittently showing 16 of 61 positive tests (26%) and 126 of 129 normal females reacted intermittently showing 135 of 512 positive tests (26%). The mean percentage of migration inhibition for all tests in patients in remission was 16.4 +/- 1.2% and that for normal controls was 7.2 +/- 0.7%; this difference was highly significant (p less than 0.001). The value for all tests in patients with relapse was 11.8 +/- 1.4%; this was statistically lower than that for patients in remission (p less than 0.05) but statistically higher than that for normal controls (p less than 0.05). A few normal women, some with high risk factors such as a strong family history and/or fibrocystic and proliferative disease, had a mean percentage of migration inhibition value in the range of that for patients with breast cancer. Mean values of sequential tests may be a more meaningful index of cellular immunity against breast cancer antigen in all groups.

Drug and hormone sensitivity of estrogen receptor-positive and -negative human breast cancer cells in vitro.

A clonogenic assay of long-term breast cancer cell cultures in vitro has been developed to provide a highly reproducible method with which to quantitate tumor cell killing by hormones and/or cytotoxic chemotherapeutic agents. Monolayer cultures of estrogen receptor-positive MCF-7 human breast cancer cells and of estrogen receptor-negative Evsa T cells are harvested by treatment with 0.01% trypsin:0.02% EDTA in Hanks' balanced salt solution. Cell suspensions are treated with drug or hormone in serum-free medium for 1 hr at 37 degrees; treated cells are washed, plated, and cultured for approximately 14 days; and colonies consisting of greater than or equal to 30 cells are counted. Compared to estrogen receptor-positive cells, estrogen receptor-negative cells were 2-fold more sensitive to melphalan but were conversely 1.9-fold more resistant to Adriamycin; these differences were statistically significant (p less than 0.001). Thus, response to cytotoxic chemotherapeutic agents appeared to be independent of estrogen receptor status. For cells treated with diethylstilbestrol, the dose of drug or hormone reducing the surviving cell fraction to 1/e (DO) for estrogen receptor-positive cells was 2.27 nmol/ml, and that for estrogen receptor-negative cells was 2.80 nmol/ml; this difference was not statistically significant. However, with tamoxifen therapy, the DO for estrogen receptor-positive cells was 0.601 nmol/ml, and that for estrogen receptor-negative cells was 3.64 nmol/ml; this 6-fold greater degree of resistance to tamoxifen of estrogen receptor-negative cells was highly significant (p less than 0.001). Treatment of cells for 24 hr with 17 beta-estradiol stimulated proliferation not only of estrogen receptor-positive cells but also of estrogen receptor-negative cells. However, estradiol at concentrations up to 200 microM had no apparent cytocidal activity, as measured by the clonogenic assay. Furthermore, treatment of MCF-7 cells simultaneously with estradiol and either diethylstilbestrol or tamoxifen failed to reverse the cytocidal activity of those two agents. These findings suggest that, in the clonogenic assay described herein, diethylstilbestrol and tamoxifen may kill human breast cancer cells by an independent mechanism of action and that the cytocidal activity of diethylstilbestrol and the proliferative effect of 17 beta-estradiol appear to be independent of estrogen receptor status.

Induction of apoptosis by deregulated expression of DNA topoisomerase IIalpha.

DNA topoisomerase II (topo II) is an essential nuclear enzyme required for chromatin condensation and chromosome segregation during mitosis. Forced overexpression of topo IIalpha was found to cause morphological changes in recipient cells associated with apoptosis. This induction of apoptosis required nuclear localization of topo IIalpha, yet was independent of the DNA cleavage-religation activity of the enzyme. Apoptosis mediated by topo IIalpha deregulation was blocked by overexpression of crmA, a specific inhibitor of certain caspases, but not by bcl-2. topo IIalpha-induced apoptosis was also blocked by overexpression of a dominant-acting mutant of stress-activated protein kinase kinase (SEK1/MKK4) but not by the overexpression of its normal counterpart. Furthermore, apoptosis was blocked by coexpression of a dominant-negative form of the cyclin-dependent kinase cdk2 but not by dominant-negative cdc2. These results provide a rationale for the tight regulation of topo IIalpha levels through the cell cycle in that deregulation of topo IIalpha expression results in apoptotic cell death.

Increased expression of cytosolic glutathione S-transferases in drug-resistant L5178Y murine lymphoblasts: chemical selectivity and molecular mechanisms.

The level of induction of three cytosolic glutathione-S-transferase (GST) classes has been compared in L5178Y murine lymphoblasts resistant to either the quinone-containing compound, hydrolyzed benzoquinone mustard (HBM), or the aromatic alkylating agent aniline mustard (AM). Three established cell lines, L5178Y/HBM2, L5178Y/HBM10, and the partial revertant, L5178Y/HBMR, were 2.5-, 6-, and 2.9-fold resistant to HBM and showed 3-, 11-, and 9-fold increases in GST activity, respectively, relative to the sensitive L5178Y cell line. Western blot analysis of cytosolic proteins showed overexpression of all three cytosolic GST classes pi, alpha, and mu, with predominance of the pi class. Northern blot analysis demonstrated corresponding elevations in the steady-state mRNA levels of each GST class. The level of GST-mu and -alpha isoforms correlated more closely with HBM resistance, whereas GST-pi, the predominant isoform in these cells, paralleled enzyme activity. These findings suggested that other factors such as quinone reductase may contribute to resistance. The AM-resistant cell line L5178Y/AM was 10-fold resistant to the alkylating agent AM, and GST activity was elevated 3.6-fold relative to the parental L5178Y cell line. Western blot analysis and Northern blot analysis provided evidence of overexpression of all three cytosolic GST classes but with marked predominance of the alpha class. These studies provide evidence that induction of GST isoforms in drug-resistant cells may have both a nonspecific as well as a selective component. The difference in isozyme profile between HBM- and AM-resistant cell lines emphasizes how structural differences, in particular, the nature of the electrophilic signal, may influence the pattern of induction of GST isozymes.

In vivo and in vitro studies of immunotherapy of nasopharyngeal carcinoma with transfer factor.

Epstein-Barr virus, the apparent cause of infectious mononucleosis, may also be an etiological agent in nasopharyngeal carcinoma and Burkitt's lymphoma. Lymphocytes from normal individuals with anti-Epstein-Barr virus antibody activity may be sensitized to Epstein-Barr virus and contain transfer factor with the potential to program and/or recruit other lymphocytes to react against the virus and/or viral antigens. A patient with nasopharyngeal carcinoma refractory to conventional therapy was treated with transfer factor obtained from normal, young adults with previous history of infectious mononucleosis. Following immunotherapy, apparent slowing of tumor growth was observed, which was associated with intense lymphocytic infiltration of the tumor and reconstitution of delayed cutaneous hypersensitivity reactions to microbial recall antigens. A double-blind randomized clinical trial has been initiated to determine whether transfer factor immunotherapy is a useful adjunct to radiotherapy in the primary treatment of patients with nasopharyngeal carcinoma. If successful, a similar trial might be considered for African patients with Burkitt's lymphoma.

Sensitivity to melphalan as a function of transport activity and proliferative rate in BALB/c 3T3 fibroblasts.

Melphalan transport by exponentially dividing BALB/c 3T3 mouse fibroblasts was approximately 3-fold greater at the steady-state than that observed with resting cells at drug concentrations ranging from 1 to 100 microM. Both the rate of incorporation of [3H]thymidine and the percentage of S-phase cells estimated by a fluorescent cell sorter provided evidence of higher DNA synthetic activity in logarithmic phase cells. Transport of melphalan by BALB/c 3T3 fibroblasts was mediated by the two amino acid transport systems, the DL-beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid-sensitive sodium-independent system preferring leucine as substrate and the sodium-dependent system preferring alanine, serine, and cysteine as substrates. At low drug concentrations (about 1 microM) the sodium-dependent mechanism predominated, but at high concentrations of melphalan (100 microM) a shift occurred from the sodium-dependent to the sodium-independent system. Both transport systems were markedly affected by the proliferative state of the cells and became progressively less active the longer cells remained in the stationary phase. Stationary-phase cells were also less sensitive to the cytotoxic action of melphalan than exponentially dividing cells. Serum deprivation experiments showed that unidirectional melphalan influx decreased about 40% when fibroblasts previously in exponential growth were shifted from normal medium to serum-depleted medium 24 h prior to drug transport studies. Thus, serum deprivation and maintenance of cells in stationary phase growth had parallel effects on drug influx. These experiments emphasize the importance of the proliferative state of the cell as a determinant of melphalan transport and cytocidal activity.

Evidence for a mutant allele of the gene for DNA topoisomerase II in adriamycin-resistant P388 murine leukemia cells.

Previous studies have shown that DNA topoisomerase II enzyme activity and protein levels are reduced in cloned lines of Adriamycin-resistant P388 leukemia cells relative to drug-sensitive cells (Deffie et al., Cancer Res., 49: 58-62, 1989). The molecular basis of the reduced topoisomerase II levels in these resistant cells has been investigated. Northern blot analysis of total cellular RNA from drug-sensitive and -resistant cells using a 1.8-kilobase human topoisomerase II complementary DNA revealed the presence of two mRNA species: a 6.6-kilobase transcript that was strongly expressed in drug-sensitive cells but reduced 7- to 8-fold in resistant cells; and a 5.5-kilobase transcript detected only in drug-resistant cells. Southern blot analysis of genomic DNA digested with BamHI, StuI, or PvuII and probed with the 1.8-kilobase complementary DNA for human topoisomerase II showed that, in Adriamycin-resistant cells, there were two different alleles for topoisomerase II, one identical to the native allele but with a lower gene copy number than that found in sensitive cells, and a second allele containing a mutation present only in resistant cells. These findings suggest that the reduced levels of topo II protein in drug-resistant cells may be due to reduced amounts of the native 6.6-kilobase mRNA. The unique 5.5-kilobase mRNA in resistant cells may represent a shortened transcript of the mutated topoisomerase II allele.

Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 leukemia cell lines.

The relationship between DNA topoisomerase II activity and drug resistance was studied in cloned cell lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia; drug resistant P388/ADR/3 (clone 3) and P388/ADR/7 (clone 7) cells are 5- and 10-fold more resistant to ADR than the sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). Topoisomerase II catalytic activity in crude nuclear extracts was reduced in drug-resistant cells as determined qualitatively by decatenation of kDNA. Using the centrifugal method fo quantitative analysis, topoisomerase II catalytic activity (mean +/- SE) was 81 +/- 10 units/mg total nuclear protein in sensitive cells, 29 +/- 2 units/mg total nuclear protein in resistant clone 3 cells, and 16 +/- 2 units/mg total nuclear protein in resistant clone 7 cells; these differences were highly significant (P less than 0.005). Similarly, quantitative analysis of DNA cleavage activity using 3' 32P-end-labeled pBR322 restriction fragments showed that drug-stimulated topoisomerase II cleavage activity in nuclear extracts from sensitive cells was approximately 1.7- and 2.9-fold greater than that from resistant clone 3 and 7 cells, respectively. Western blot analysis of nuclear extracts from the three cell lines using antibody against the C-terminal half of recombinant-prepared human topoisomerase II polypeptide revealed reduced immunoreactivity of topoisomerase II protein in the drug-resistant cells. These data suggest that reduced topoisomerase II activity in resistant cells, which may represent quantitative reduction of the enzyme, may be another property contributing to multifactorial drug resistance in these cells.

Comparison of adriamycin uptake in chick embryo heart and liver cells an murine L5178Y lymphoblasts in vitro: role of drug uptake in cardiotoxicity.

The mechanism of uptake of Adriamycin was investigated in chick embryo heart and liver cells and in murine L5178Y lymphoblasts in vitro. Drug uptake at 4 degrees C for 15 s due to rapid association accounted for a cell:medium distribution ratio of 104 +/- 14 (SE) in heart cells, 10.2 +/- 1.3 in liver cells, and 10.3 +/- 1.5 in L5178Y lymphoblasts. On thin-layer chromatographic analysis, 98% of the radioactivity migrated with the mobility of intact drug, suggesting that drug metabolism was negligible for at least 30 min in both heart and tumor cells. A time course of drug uptake was somewhat different in heart cells compared to that noted for liver or L5178Y cells. The steady state for drug uptake was reached more promptly in heart cells; apparent equilibrium was observed at 6 min in heart cells, at approximately 20 min in L5178Y lymphoblasts, but was not attained by 25 min in liver cells. Temperature dependence of drug uptake also differed in the three cell types; drug uptake was most temperature sensitive in L5178Y cells, intermediate in liver cells, and least temperature dependent in heart cells. Separation of heart, liver, and leukemic cells into membrane and cytosol fractions demonstrated that, at 1 and 30 min, more than 75% of the drug was associated with the membrane fraction. Trichloroacetic acid extraction of cell constituents revealed that, at 1 min, the acid-soluble fraction amounted to 32 +/- 2% of radioactivity in heart cells and 37 +/- 2% in L5178Y cells. Ethanol extraction of these cells demonstrated that, at 1 min, ethanol-soluble components accounted for 49 +/- 2% of radioactivity in heart cells and 27 +/- 2% in leukemic cells. The finding of a large component of rapid association together with evidence of prompt drug binding to cellular constituents made evaluation of unidirectional drug influx impractical. Accordingly, an investigation was undertaken of Adriamycin efflux from chick embryo heart and liver cells and L5178Y lymphoblasts, after the cells had been loaded with drug for various time intervals. In all three cell types, efflux was rapid down to a plateau level, representing nonexchangeable drug. As the period of time for loading cells was increased, there was a progressive rise in the level of nonexchangeable drug. Equilibration of the nonexchangeable pool occurred more rapidly in heart cells than in either liver or leukemic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a DNA topoisomerase IIalpha gene rearrangement in adriamycin-resistant P388 leukemia: expression of a fusion messenger RNA transcript encoding topoisomerase IIalpha and the retinoic acid receptor alpha locus.

Previous studies using cloned lines of Adriamycin-sensitive and -resistant P388 murine leukemia cells have suggested that a reduction in DNA topoisomerase II alpha (topo II alpha) enzyme activity and protein levels in drug-resistant cell lines (A. M. Deffie, J. K. Batra, and G. J. Goldenberg, Cancer Res., 49: 58-62, 1989) may be due to an allelic mutation in the topo II alpha gene (A. M. Deffie, D. J. Bosman, and G. J. Goldenberg, Cancer Res., 49: 6879-6882, 1989). The drug-resistant cell lines P388/ADR/3 and P388/ADR/7 express a shortened topo II alpha mRNA transcript in addition to the native transcript present in the drug-sensitive P388/4 cell line. Using complementary DNA probes derived from the coding sequence and 3' untranslated region of the native mouse topo II alpha transcript, we have determined that the shorter 4.5-kilobase topo II alpha transcript expressed in the drug-resistant cell lines contains only 3.5-kilobases of topo II sequence from the 5'-terminus onwards. Using a 3'-rapid amplification of cDNA ends strategy, we have cloned cDNAs representing the 3'-termini of both the native and mutant transcripts from both P388/ADR/3 and P388/ADR/7 cells. DNA sequence analysis revealed that the shorter 4.5-kilobase transcript: (a) encodes topoisomerase II alpha until nucleotide position 3494, at which point the sequence diverges for the remaining 956 bases; (b) contains a polyadenylation signal distinct from the native transcript; and (c) contains an open reading frame predicting a truncated topo II alpha fusion protein. Of great interest was the finding that the non-topo II alpha 956-base sequence in the shorter transcript encodes the promoter, exon I, and part of the first intron of the murine retinoic acid receptor alpha gene locus in the antisense orientation, suggesting that a rearrangement on chromosome 11 in the drug-resistant cells led to a gene fusion event between the loci encoding topo II alpha and retinoic acid receptor alpha.

Mechanism of uptake of nitrosoureas by L5178Y lymphoblasts in vitro.

The mechanism of uptake of nitrosoureas by L5178Y cells in vitro was investigated. A time course of the uptake of radioactivity on incubation of L5178Y lymphoblast with [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea was linear for 30 min and then entered a plateau phase; it was markedly temperature dependent. A similar time course for cells incubated with [14C]ethylene-labeled 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea reached equilibrium rapidly, was temperature independent, and resulted in a relatively low level of uptake of radioactivity. However, cells treated with 3-[cyclohexyl-14C]-1-(2-chlorethyl)-1-nitrosourea had a time course that was linear for 30 min, resulted in much higher levels of uptake of radioactivity, and was strongly temperature dependent. These findings, at least for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, suggest that some drug decomposition precedes uptake. The percentage of radioactivity found in the cell sap fraction was at least 85% of total cell activity when cells were incubated with any of the three 14C-labeled nitrosoureas. Furthermore, thin-layer chromatography of the cell sap fraction revealed the presence of free intact drug. These findings indicate that intracellular uptake of intact nitrosoureas occurred. A time course of uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea reached equilibrium rapidly with cell/medium distribution ratios of 0.2 to 0.6 and was temperature independent. The addition of excess unlabeled 1,3-bis(2-chlorethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea had no effect on uptake of [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea, These findings suggest that uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea was by passive diffusion. A time course of the uptake of intact 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with either [14C]ethylene- or ring-labeled drug rapidly reached equilibrium, was temperature independent, and attained a cell/medium ratio greater than unity. Uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was sodium independent and was unaffected by the metabolic inhibitors (sodium fluoride, sodium cyanide, or 2,4-dinitrophenol) or by urea, a potential physiological competitor. Furthermore, addition of unlabeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or 1,3-bis(2-chlorethyl)-1-nitrosourea had no effect on uptake of labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. These findings suggest that uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea also occurs by passive diffusion.

Resistance to adriamycin: relationship of cytotoxicity to drug uptake and DNA single- and double-strand breakage in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia.

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established from single cell cultures. A marker chromosome M1 was found in all cells in the heterogeneous resistant P388/ADR parental line as well as in the cloned resistant lines P388/ADR/3 and P388/ADR/7; a different marker chromosome M2 was present in the heterogeneous sensitive P388 parental line as well as the cloned sensitive line P388/4. Dose-survival studies showed that D0, the dose of Adriamycin reducing survival to 1/e (i.e., 37% of the initial population), was 33 +/- 5 (SE) nM for sensitive P388/4 cells, 169 +/- 17 nM for resistant P388/ADR/3 cells, and 336 +/- 28 nM for the more resistant P388/ADR/7 cells. Drug uptake in sensitive P388/4 cells was 1.6-fold greater than in resistant P388/ADR/3 cells and 2.1-fold greater than in resistant P388/ADR/7 cells. The number of DNA single-strand breaks produced per microM Adriamycin was 131 +/- 9 rad equivalents in sensitive clone 4 cells, 41 +/- 8 rad equivalents in resistant clone 3 cells, and 33 +/- 11 rad equivalents in resistant clone 7 cells. The number of DNA double-strand breaks per microM Adriamycin was 1721 +/- 126 rad equivalents in sensitive cells, 117 +/- 36 rad equivalents in resistant P388/ADR/3 cells, and 194 +/- 16 rad equivalents in resistant P388/ADR/7 cells. Differences in drug uptake were insufficient to explain the higher incidence of DNA single- and double-strand breaks in sensitive cells. These findings strongly support the concept that resistance to Adriamycin in P388 leukemia cells is multifactorial; however, this study did not resolve whether these changes arise from a single pleiotropic mutation or from multiple mutations. In sensitive P388/4 cells the number of DNA single-strand breaks formed could all be attributed to double-strand breaks. However, in both resistant cell lines the level of induction of single-strand breaks was in excess of that due to double-strand breaks, and this excess of single-strand breaks appeared to vary directly with the degree of resistance, being greater in the more resistant clone 7 cells than in the less resistant clone 3 cells. In both sensitive and resistant cell lines the ratio of true single- to double-strand breaks varied inversely with the concentration of Adriamycin. Finally, the cytotoxic activity of Adriamycin appeared to correlate more closely with formation of DNA double-strand breaks than with single-strand lesions.

Mechanism of efflux of melphalan from L5178Y lymphoblasts in vitro.

The mechanism of efflux of melphalan from L5178Y lymphoblasts in vitro was investigated. Evidence was obtained that drug efflux occurs by a process different from the influx mechanism. A time course of efflux followed a first-order process for at least 5 min, with a rate constant K of 0.13 +/- 0.05 (S.D.) min-1, and approximately 80% of drug taken up by the cells was exchangeable. From a kinetic analysis of melphalan efflux, it was not possible to determine if the mechanism of drug efflux was simple diffusion or a technically nonsaturable carrier-mediated process. The Q10 for drug efflux varied from 1.2 to 1.8, values intermediate between those expected for simple diffusion and a carrier-mediated process. Furthermore, drug efflux was sodium independent and was not inhibited by the presence of amino acids on the same side of the membrane, findings which support the concept that efflux was by simple diffusion. The presence of a wide variety of amino acids in the extracellular medium was found to stimulate melphalan efflux. An evaluation of the structure-activity relationship disclosed that amino acids containing hydroxyl, acidic, or amide side chains were most active; however, a distinct pattern between amino acid structure and stimulation of efflux was not established. The stimulation process was concentration dependent and was not restricted to either L5178Y cells or to melphalan as transport substrate.

Uptake and decomposition of chlorozotocin in L5178Y lymphoblasts in vitro.

Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled chlorozotocin. A time course of uptake of total radioactivity revealed a greater cell/medium distribution ratio of activity in cells treated with chloroethyl-labeled chlorozotocin compared to cells treated with the glucose-labeled compound. Thin-layer chromatographic analysis showed that uptake of intact chlorozotocin was identical in cells treated with either glucose- or chloroethyl-labeled drug and that the cell/medium distribution ratio never exceeded unity. Accumulation of 14C-chlorozotocin was not inhibited by an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, or several metabolic inhibitors or by sodium ion depletion. These observations, together with the relatively low temperature quotient for the uptake process, suggested that chlorozotocin uptake occurs by passive diffusion. In cells treated with glucose-labeled chlorozotocin, a bicyclic urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-labeled drug, nonpolar as well as polar metabolites were noted. Formation of metabolites from the glucose moiety was impeded by the presence of an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, the glucose transport inhibitors phlorizin and phloretin, the metabolic inhibitor m-chlorophenyl carbonyl cyanide hydrazone and by sodium depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chlorophenyl carbonyl cyanide hydrazone and by sodium ion depletion. These results suggested that metabolism of chlorozotocin in L51789Y lymphoblasts appears to be enzyme mediated.

Growth-promoting activity of desmopressin in murine leukemia cells treated in vitro.

The synthetic vasopressin analogue, desmopressin (dDAVP), has been shown to influence membrane transport of melphalan in murine L5178Y lymphoblasts. Accordingly, the effect of dDAVP on the cytocidal activity of melphalan in L5178Y cells was evaluated. dDAVP did not affect the cytocidal activity of melphalan in these cells, but significantly affected the cloning efficiency of stationary phase or slowly dividing L5178Y cells over a range of concentrations. In particular, stationary phase cells showed an increase in cloning efficiency from 4.3 +/- 0.5% in control cells to 7.0 +/- 0.3% in cells treated with 25 nM dDAVP (P less than 0.001), whereas cells doubling every 26 h showed an increase from 10.8 +/- 1.2% in control cells to 21.0 +/- 2.0% in cells treated with 150 nM dDAVP (P less than 0.001). This phenomenon was associated with significant elevations of 1,2[3H] diacylglycerol after incubation with dDAVP for 9 min (P less than 0.01) and of total [3H]diacylglycerols after incubation for both 3 min (P less than 0.05) and 9 min (P less than 0.02). Within 10 s of treatment with 100 nM dDAVP, there was a marked decrease in the levels of inositol 1,4,5-trisphosphate and inositol 1-phosphate, but subsequently no change was observed for up to 9 min after treatment. We postulate that the increase of diacylglycerol content produced by dDAVP might be primarily from a phosphatidylcholine source and that the growth-promoting activity of desmopressin may be a consequence of activation of protein kinase C.

Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status.

We have previously reported the identification and characterization of a prolactin-inducible protein (PIP) as well as the cloning of the gene encoding PIP in cultured human breast cancer cells. We now present three lines of evidence that the gene encoding PIP is also expressed by some human breast cancers in vivo: detection of PIP immunoreactivity in the serum of some breast cancer patients; immunohistochemical detection of PIP in breast cancer sections; and the presence of PIP mRNA, detected by complementary DNA hybridization in human breast biopsy samples. In a preliminary study using Western blot analysis authentic PIP was detected in the serum of some patients with breast cancer. Subsequently the sera of 234 unselected patients with breast cancer were assayed for the presence of PIP using a specific radioimmunoassay. Thirty-five % of these sera contained detectable PIP (i.e., greater than 3 ng/ml). As well as were able to show by immunohistochemical techniques that PIP immunoreactivity was present in some human breast biopsy specimens. Levels of estrogen receptor, progesterone receptor, and PIP mRNA were determined in an unselected population of 51 human breast tumor biopsies. Sixty-one % of these tumors had detectable PIP mRNA; a positive correlation (r = 0.52; P less than 0.01) was found between PIP mRNA levels in breast biopsy samples and estrogen receptor content, a known prognostic indicator in human breast cancer.