Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Phase I and pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2 weeks in patients with advanced solid tumors.

BACKGROUND: Lexatumumab (HGS-ETR2) is a fully human agonistic mAb to the tumor necrosis factor-related apoptosis-inducing ligand receptor 2 that activates the extrinsic apoptosis pathway and has potent preclinical antitumor activity. MATERIALS AND METHODS: This phase 1, dose escalation study assessed the safety, tolerability, pharmacokinetics (PKs) and immunogenicity of lexatumumab administered i.v. every 14 days in patients with advanced solid tumors. RESULTS: Thirty-one patients received lexatumumab over five dose levels (0.1-10 mg/kg). Most (26 of 31) received four or more cycles of treatment. One patient at 10 mg/kg experienced a possibly related dose-limiting toxicity of grade 3 hyperamylasemia. Nine patients achieved stable disease. One patient with chemotherapy-refractive Hodgkin's disease experienced a mixed response. Lexatumumab PKs were linear up to 10 mg/kg. At the 10 mg/kg dose, the mean (+/-standard deviation) t(1/2b) was 13.67 +/- 4.07 days, clearance was 4.95 +/- 1.93 ml/day/kg, V(1) was 45.55 ml/kg and V(ss) was 79.08 ml/kg, indicating that lexatumumab distributes outside the plasma compartment. No human antihuman antibodies were detected. CONCLUSIONS: Lexatumumab can be safely administered every 14 days at 10 mg/kg. The PK profile supports this schedule. Further evaluation of lexatumumab at this dose schedule is warranted, including combination trials with other agents.

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Dissociation of antitumor potency from anthracycline cardiotoxicity in a doxorubicin analog.

The search for new congeners of the leading anticancer drug doxorubicin has led to an analog that is approximately 1000 times more potent, noncardiotoxic at therapeutic dose levels, and non-cross-resistant with doxorubicin. The new anthracycline, 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN), is produced by incorporation of the 3' amino group of doxorubicin in a new cyanomorpholinyl ring. The marked increase in potency was observed against human ovarian and breast carcinomas in vitro; it was not accompanied by an increase in cardiotoxicity in fetal mouse heart cultures. Doxorubicin and MRA-CN both produced typical cardiac ultrastructural and biochemical changes, but at equimolar concentrations. In addition, MRA-CN was not cross-resistant with doxorubicin in a variant of the human sarcoma cell line MES-SA selected for resistance to doxorubicin. Thus antitumor efficacy was dissociated from both cardiotoxicity and cross-resistance by this modification of anthracycline structure.

Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro.

Human small cell lung carcinoma (SCLC) cells have been shown to contain significant levels of a bombesin-immunoreactive peptide. The 27-amino acid peptide, gastrin releasing peptide (GRP), has recently been shown to be responsible for the bombesin-like immunoreactivity found in SCLC cells. Among four lung cancer cell lines examined in vitro, GRP exhibited mitogenic activity for two SCLC subtypes, but not for a squamous carcinoma or adenocarcinoma lung cell line. The mitogenicity of the GRP molecule has been isolated to the carboxyterminal fragment, designated GRP 14-27, which is in part homologous to bombesin. The aminoterminal fragment, GRP 1-16, is no homologous to bombesin and exhibits no mitogenic activity. Thus, GRP may be an important growth regulating or autocrine factor in human SCLC.

Low or absent SPARC expression in acute myeloid leukemia with MLL rearrangements is associated with sensitivity to growth inhibition by exogenous SPARC protein.

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to S phase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

Characterization of covalent DNA binding of morpholino and cyanomorpholino derivatives of doxorubicin.

BACKGROUND: The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activation in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation. Alkylation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs. PURPOSE: Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross-linking DNA. METHODS: We used [3H]MRA and [3H]MRA-CN, with the 3H labeled at C-2 and C-6 of the morpholino ring. MRA (10 nM) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcinoma cell line ES-2. We studied the appearance of interstrand cross-links versus total DNA adducts in pBR322 plasmid DNA incubated with 100 nM MRA-CN in cell-free medium and then subjected to denaturation and agarose gel electrophoresis. RESULTS: Regardless of the extracellular concentration of the drug (1-100 nM), 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 nM MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA if the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentage of plasmids containing at least one interstrand cross-link rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecules of MRA-CN were adducted per molecule of pBR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross-links. CONCLUSIONS: These results suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross-links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA.

DNA cross-linking and cytotoxicity of the alkylating cyanomorpholino derivative of doxorubicin in multidrug-resistant cells.

The cyanomorpholino derivative of doxorubicin (MRA-CN) is an anthracycline that is extremely potent and non-cross-resistant with doxorubicin (DOX) in multidrug-resistant cells. MRA-CN binds to and cross-links DNA and thus has been proposed to act as a targeted alkylating agent. In our study, the number of DNA interstrand and DNA-protein cross-links produced by MRA-CN was identical in multidrug-resistant Dx5 and parental MES-SA cells, as shown by alkaline elution analysis. The amount of cross-linking was directly proportional to drug concentration at concentrations from 10(-11) to 10(-7) M MRA-CN. Extensive DNA cross-linking was evident within 30 minutes of drug exposure. After 1 hour of drug exposure, the number of DNA cross-links increased for 90 minutes, reached a plateau, and then began to decrease after 120 minutes. Loss of cell viability was also observed as early as 3 hours after exposure to MRA-CN. The finding of the same number of DNA cross-links in MES-SA and Dx5 cells indicates that similar amounts of MRA-CN are likely to enter the nuclei of multidrug-resistant and sensitive cells. Other anthracyclines have major differences in nuclear distribution in sensitive and resistant cells. Several factors may contribute to the non-cross-resistance of MRA-CN in multidrug-resistant cells. (a) The lipophilicity of MRA-CN facilitates cell entry. (b) The substitution and loss of basicity at the amino nitrogen may reduce the affinity of the drug for the P-glycoprotein efflux pump, compared with that of DOX. (c) The detoxification function of P-glycoprotein may be less effective for drugs that produce rapid and irreversible cell damage, such as the DNA-targeted alkylation caused by MRA-CN.

Association of DNA cross-linking with potentiation of the morpholino derivative of doxorubicin by human liver microsomes.

The morpholino analog of doxorubicin (DOX), 3'-deamino-3'-(4"-morpholinyl)-doxorubicin (MRA), is 0.5- to 10-fold more potent than DOX in vitro but 100- to 200-fold more potent in vivo, which indicated that biotransformation in vivo may generate a highly potent metabolite(s). A likely mechanism for such biotransformation is hepatic mixed-function oxidation. At a concentration of 5 microM, MRA was incubated for 30 minutes at 37 degrees C with 1 mg of human liver microsomes/mL and 0.45 mM of NADPH. The cytotoxicity of the microsome- and NADPH-treated MRA was 44-fold higher than that of the untreated MRA in the human ovarian carcinoma cell line ES-2. This potentiation did not occur for MRA treated with boiled microsomes and NADPH, active microsomes in the absence of NADPH, or Tris buffer plus NADPH. No potentiation was observed with DOX or the highly potent cyanomorpholino derivative of DOX, MRA-CN, under any of the above conditions. After 2 hours of exposure of the ES-2 cells to microsome- and NADPH-treated MRA, dose-dependent DNA cross-links were observed with 5 nM or more of MRA, whereas only DNA strand breaks were detected in cells exposed to 500 nM of untreated MRA or MRA incubated under other conditions. These data indicate that MRA is biotransformed by the hepatic mixed-function oxidases to a potent DNA-alkylating metabolite(s), which may be important in the determination of the pharmacologic and toxicologic profile of MRA. The active metabolite(s) of MRA may be analogous to MRA-CN, which cross-links DNA without requiring bioactivation.

Doxorubicin and the alkylating anthracycline 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin: comparative in vitro potency against leukemia and bone marrow cells.

The new anthracycline analogue 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin (MRA-CN) is an intensely potent compound that has been shown to be 100-1,000 times more potent than doxorubicin (DOX) in vivo and in vitro. In addition, MRA-CN has been non-cross-resistant with DOX in DOX-selected models of multidrug resistance. We now report the effect of MRA-CN (and DOX) on leukemia cell lines established from patients with common, T-cell, and B-cell acute lymphoblastic leukemia, as well as with monoblastic leukemia. The effect of MRA-CN on the leukemia cells was compared to its toxicity on normal myeloid progenitors (therapeutic ratio) and to the effect of DOX on the leukemia and normal cells. MRA-CN was found to be 100 times more potent than DOX against normal myeloid progenitors--colony-forming units, granulocyte-macrophage (CFU-GM)--and 40-240 times more potent than DOX against leukemia cell lines. In addition, the therapeutic ratio was uniformly greater than 1, indicating that each leukemia cell line tested was more sensitive than CFU-GM to MRA-CN in vitro. There was a lack of correlation between MRA-CN and DOX at a drug concentration at which the colony formation is inhibited by 50% in the leukemia cell lines (correlation coefficient = 0.38), which supported the previous reports of non-cross-resistance between these two agents. The favorable therapeutic ratio, the non-cross-resistance with DOX, and the previously described lack of cardiac toxicity all make MRA-CN an attractive candidate for clinical trials in patients with acute leukemia.

Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis.

BACKGROUND: The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes. There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells. PURPOSE: We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones. METHODS: Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask). After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C). Surviving colonies in each of the 30 populations were scored and individually harvested. RESULTS: Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation. Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B. Five of the stably resistant clones were cross-resistant to doxorubicin. Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones. No increase in expression of the MRP gene was observed. However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants. CONCLUSIONS: Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance. The experimental design has derived a set of resistant mutants from a single-step selection. In those clones, decreased expression of Topo II is the predominant mechanism selected. IMPLICATIONS: These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure. The stably resistant clones may represent descendants from a single mutational event in each population.

Decreased mutation rate for cellular resistance to doxorubicin and suppression of mdr1 gene activation by the cyclosporin PSC 833.

BACKGROUND: Various mechanisms can contribute to cellular resistance to doxorubicin. These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene [also known as PGY1], Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism, and altered levels or activity of topoisomerase II (Topo II). We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation. All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene. PURPOSE: Luria and Delbrück fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance. METHODS: Ten flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells. After reseeding in 96-well plates, the populations were treated with 40 nM doxorubicin and 2 microM PSC 833 for 3 weeks. Surviving colonies were scored, individually harvested, and propagated. The drug-resistant phenotype was assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation. Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression. Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry. Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay. RESULTS: Mutations were detected at a rate of 2.5 x 10(-7) per cell generation. Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833. None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression. No alterations were found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts. However, a significant decrease in Topo II alpha messenger RNA and protein was found in all examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide. CONCLUSIONS: PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants. Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression. IMPLICATIONS: Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.

Multidrug (pleiotropic) resistance in doxorubicin-selected variants of the human sarcoma cell line MES-SA.

The emergence of drug-resistant tumor cells is a major limiting factor in cancer chemotherapy. There is little information about the nature of such resistant variants among human cancer cell populations. Doxorubicin (DOX)-resistant sublines of the human sarcoma cell line MES-SA were selected by continuous in vitro exposure to DOX. Stepwise increases in DOX concentration produced variants which were 25- and 100-fold resistant to DOX. These sublines displayed marked cross-resistance to daunorubicin, dactinomycin, mitoxantrone, colchicine, vincristine, vinblastine, and etoposide and moderate resistance to mitomycin C and melphalan. Cross-resistance was not observed, however, to methotrexate, 5-fluorouracil, bleomycin, carmustine, or cisplatin. DOX resistance in these cell lines appeared to be stable despite long periods of growth in drug-free medium. Two additional marker chromosomes were identified in the 100-fold resistant variant, which indicated clonal selection during drug exposure, but no double minute chromosomes or homogeneously staining regions were noted. Doxorubicin accumulation in the DOX-resistant cells was reduced by approximately 50% compared to that of the sensitive MES-SA cells, as a result of enhanced efflux of DOX from the resistant cells. There was no evidence of appreciable DOX metabolism by either the sensitive or resistant cells. These studies demonstrate marked DOX resistance and multidrug resistance arising in a human sarcoma line during exposure to DOX. The pleiotropic nature of this resistance is similar to that described in other models. Decreased drug accumulation due to enhanced drug efflux is identified as a major mechanism of resistance in these cells, although other factors may also be involved.

Reversal by cefoperazone of resistance to etoposide, doxorubicin, and vinblastine in multidrug resistant human sarcoma cells.

The cephalosporins are a family of semisynthetic antibiotics, some of which have structural features associated with substrates for the multidrug transporter, P-glycoprotein. The activity of a series of six cephalosporins in reversing multidrug resistance (MDR) was examined in MDR variants (Dx5 cells) of the human sarcoma line MES-SA. Dx5 cells express high levels of the mdr1 gene product P-glycoprotein and are 25- to 30-fold resistant to doxorubicin (DOX), etoposide (VP-16), and vinblastine (VBL). Cytotoxicity was measured by the MTT assay. Cefoperazone (1.0 mM) was the most effective modulator of MDR, lowering the IC50 for VP-16 by 29-fold (29x), for VBL by 16x, and for DOX by 14x. Ceftriaxone at 1.0 mM produced 10x modulation of VP-16 cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of resistance was concentration dependent, decreasing to 4x and 5x, respectively, for DOX with 0.25 mM cefoperazone and ceftriaxone. No modulation of cytotoxicity was observed in the parental MES-SA cells, which do not express mdr1. Cefazolin, cefotetan, cephradine, and ceftazidime were ineffective, producing less than 5x modulation of DOX at 1.0 mM. Among these cephalosporins, cefoperazone and ceftriaxone were the most highly protein bound in the media (30 and 52%), and the most lipid soluble, with octanol/water partitioning coefficients of -0.49 and -0.60. Varying the serum concentration in medium from 5 to 50% had less than a two-fold effect on the modulation of MDR by ceftriaxone. The ability to reverse MDR among these agents is associated with lipid solubility, high protein binding, a polycyclic planar geometry, and the presence of the piperazine group in cefoperazone. These data and the potential for achieving high tissue concentrations indicate that cefoperazone merits further study as a modulator of MDR.

Development and characterization of a human sarcoma cell line, MES-SA, sensitive to multiple drugs.

A cell line designated MES-SA has been developed from a uterine sarcoma. Cells from the surgical tumor specimen were grown in a soft-agar clonogenic assay, with a relatively high plating efficiency of 0.5% and sensitivity to multiple drugs. Histologically, the surgical specimen and tumors developing after MES-SA inoculation into nude mice were identical, consisting of sheets of anaplastic sarcoma cells amid scant hyalinized stroma. The nonepithelial origin of this line was supported by ultrastructural analysis and negative mucin staining. Growth in monolayer was established by seeding colonies from soft agar into liquid media and has been maintained for over 21 months (greater than 100 passages), with a population-doubling time for the cell line of 22 hr. The MES-SA line readily forms colonies in soft agar with plating efficiencies ranging from 10 to 20%. Tumor cell inoculation s.c. into nude mice produces tumors within 2 to 3 weeks and subsequent tumor volume-doubling times of 7 to 10 days. MES-SA has a modal chromosome number of 45. Karyotypic abnormalities include: monosomic forms of chromosomes 5, 6, and 7; a 5q, 6p translocation; and one marker chromosome. In vitro sensitivities to doxorubicin, dactinomycin, mitomycin C, and bleomycin have been demonstrated by clonogenic assay. These drug sensitivities remain stable over long periods of monolayer growth and after passage in nude mice.

Enhancement of bleomycin activity against Lewis lung tumors in mice by local hyperthermia.

The cytotoxicity of the drug bleomycin in vitro has previously been shown to be enhanced by hyperthermia. This report demonstrates in vivo a more than additive interaction between local tumor hyperthermia (43 degrees, 60 min) and bleomycin (15 mg/kg s.c.) against s.c.-implanted Lewis lung carcinomas in mice. Local hyperthermia was produced by the application of 2450-MHz microwaves to the region of the tumor without induction of significant whole-body hyperthermia. The combined drug and heat treatments were applied to tumors on Days 4, 7, and 10 following implantation. The response of the tumors to simultaneous treatment was a 17-day growth delay compared with controls, whereas the local hyperthermia and bleomycin individually resulted in only 3- and 4-day growth delays, respectively. If the two treatments were given either 4 or 24 hr apart only an additive effect on growth delay was observed.

Paradoxical increase in DNA cross-linking in a human ovarian carcinoma cell line resistant to cyanomorpholino doxorubicin.

The cyanomorpholino analog of doxorubicin (MRA-CN) is a potent cytotoxic agent which is known to cross-link DNA. A human ovarian carcinoma cell line, ES-2, was grown in increasing concentrations of MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to MRA-CN. DNA damage and repair in response to MRA-CN were compared in the parental and resistant cell lines using alkaline elution. DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of MRA-CN. Non-protein-associated DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent DNA cross-linking observed with the ES-2R cells after exposure to MRA-CN. Studies of the time course of cross-link repair after exposure to MRA-CN revealed that 75% of the DNA cross-links disappeared in the ES-2R cells by the end of 8 h in drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in DNA strand breaks. The topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However, proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by MRA-CN, suggesting that topoisomerase II may not be involved. These findings indicate that, in addition to DNA cross-linking, MRA-CN causes DNA strand breakage. Resistance to MRA-CN in the ES-2R cells is associated with more apparent DNA cross-linking and less DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific DNA degradation between the resistant and the sensitive cell lines.

Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin.

The cyanomorpholino derivative of doxorubicin (MRA-CN) is a DNA intercalator and alkylator that is a highly potent cytotoxin, non-cross-resistant in multidrug-resistant cells, and noncardiotoxic in comparison with doxorubicin. To further examine mechanisms of action and resistance to MRA-CN, a cell line resistant to MRA-CN, ES-2R, was established by growing a human ovarian carcinoma cell line, ES-2, in increasing concentrations of the drug. The resistant subline was 4-fold resistant to MRA-CN and cross-resistant to other DNA cross-linking agents, cisplatin (7-fold) and carmustine (3-fold), as well as to the DNA strand-breaking agents etoposide (6-fold), doxorubicin (2-fold), bleomycin (5-fold), and ionizing radiation (2-fold). In contrast, ES-2R cells were not cross-resistant to vinblastine. Several months of additional growth of ES-2R cells in MRA-CN did not yield higher, stable levels of drug resistance. A low level of P-glycoprotein was detectable in the ES-2R cells. However, the extent of intracellular accumulation of [3H]MRA-CN by this resistant cell line was identical to that of the sensitive line. The number of DNA cross-links formed by cisplatin in ES-2R was only 50% of that of the ES-2 cells and was associated with a 50% increase in the rate of repair of these cross-links in the resistant cells. Ionizing radiation induced similar amounts of single- and double-strand breaks in the ES-2 line as well as in the ES-2R cells. There was no apparent difference between the two cell lines in the rate and extent of repair of these DNA breaks. Thus, enhanced DNA repair cannot explain the phenomenon of cross-resistance to radiation. Comparisons of glutathione (GSH) content and the enzymes involved in GSH homeostasis showed significant differences. Resistant cells contained 1.5-fold more GSH, a 2.2-fold increase in gamma-glutamyltranspeptidase activity, and a 2.4-fold increase in GSH reductase compared with ES-2 cells (all P less than 0.05). Total glutathione-S-transferase (GST) activity was 2.6-fold higher (P less than 0.01) in the ES-2R line. The pi-class GST subunit by Western blotting and GST activity toward ethacrynic acid were increased 2-fold in the resistant cells. Depletion of GSH levels in ES-2R cells by buthionine sulfoximine restored the sensitivity of ES-2R to MRA-CN. These findings implicate a role for GSH metabolism in the resistance phenotype of ES-2R cells. We have previously reported that these cells have an increased generation time and decreased topoisomerase II content. Thus, the ES-2R cell line exhibits a complex phenotype of broad cross-resistance, which is likely to involve multiple mechanisms, and includes enhanced DNA repair and increased GSH content and GST activity.

Antibody-directed targeting of liposomes to human cell lines: role of binding and internalization on growth inhibition.

Small unilamellar liposomes containing methotrexate or methotrexate-gamma-aspartate were conjugated to Staphylococcus aureus protein A and were thus able to bind cell-specific immunoglobulins for targeting to malignant human B- and T-cell lines. We were able to demonstrate enhanced protein A liposome uptake and growth inhibition by targeting with an anti-major histocompatibility complex class II antibody recognizing two different B-cell lines. The enhanced growth inhibition was specific for the targeting antibody and amounted to a 2- to 3-fold lowering of the concentration of drug required to inhibit cell growth by 50% as compared to nontargeted liposomes or liposomes targeted with an antibody not recognizing a cell surface antigen. A strong association between enhanced growth inhibition and liposome internalization as assessed by fluorescent-activated cell sorter analysis of carboxyfluorescein containing protein A liposomes was seen. By contrast, specific enhancement of growth inhibition was not seen with several anti-idiotype antibodies or antibodies to T-cell differentiation antigens. Liposome internalization did not occur with these antibodies. Failure of growth inhibition and PA liposome internalization could not be explained by differences in cell binding of the antibody PA liposomes or the degree of protein A binding of the targeting antibody. Although the ability of the targeting antibody to bind to the cell and to protein A are important, these factors alone are not sufficient to guarantee internalization and growth inhibition. Variations in rates of internalization of various cell surface antigen-antibody complexes may account for different protein A liposome mediated cytotoxicities.

Characterization and immunoassay of human tumor-associated galactosyltransferase isoenzyme II.

Galactosyltransferase (GT) (EC 2.4.1.38) was purified to homogeneity from human ovarian tumor effusion fluid and normal human serum by chromatography on alpha-lactalbumin and anti-human immunoglobulin affinity (to selectively absorb contaminating IgG) columns. Both preparations showed a single, broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis centered at a molecular weight of 48,000, but nondenaturing polyacrylamide gel electrophoresis of GT isolated from tumor effusion fluid revealed the presence of a series of oligomeric proteins possessing GT activity, which were barely detectable in normal human serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of N-glycanase- and O-glycanase-treated GT revealed that each endoglycanase removed carbohydrate with an approximate molecular weight of 3,000, revealing the presence of both N-linked and O-linked oligosaccharide substitutions on GT. Purified GT (containing a mixture of GT isoenzymes) was used to immunize BALB/c mice for monoclonal antibody (MAb) preparation. Four of the MAb isolated reacted with GT. MAb 3872 (patent pending; an IgG1) was determined to be specific for a cancer-associated GT isoenzyme (GT-II) by immunostaining of Western blots and nondenaturing polyacrylamide gel electrophoresis of GT specifically eluted from a MAb 3872 affinity column. Two 125I-labeled cyanogen bromide peptides (Mr 8,400 and 7,400) prepared from 125I-GT were specifically bound and eluted from a MAb 3872 affinity column, demonstrating that the MAb 3872 GT-II-specific antigenic epitope resides on these peptides. MAb 3872 was immobilized on 1,1'-carbonyldiimidazole-activated trisacryl GF-2000 and used to specifically assay serum GT-II levels in 29 individual normal human serum samples and 77 serum samples from 38 patients with advanced ovarian tumors. The normal serum GT-II level was found to be 85.3 +/- 30.9 milliunits/ml, with a range of 17 to 160 milliunits/ml. Of the 38 tumor patients, 33 showed GT-II values in excess of 200 milliunits/ml, with a range of 216 to 8,469 milliunits/ml. Serial samples obtained from the ovarian tumor patients suggested that the serum GT-II level reflected the tumor burden of the patient.