Nrf2-Regulated Antioxidant System in Cells of In Vivo Drug-Resistant P388 Murine Leukemia Strains.

We studied the expression of Nrf2 transcription factor and antioxidant system proteins in drug-resistant murine leukemia strains P388 in vivo, as well as the redox status of cells under conditions of induced oxidative stress. Immunoblotting and real-time PCR showed that the cyclophosphamide-resistant strain P388 (P388/CP) exhibits Nrf2-mediated drug resistance. Cells of the P388/CP strain are characterized by high expression of Nrf2, which leads to a significant increase in the expression of ARE genes and antioxidant system proteins, as well as to the effective maintenance of redox homeostasis under conditions of induced oxidative stress. Taking into account the important role of Nrf2 overexpression in reducing the effectiveness of chemotherapy in patients with different leukemias, the P388/CP strain can be of great interest as a model in the development of new drugs for the treatment of malignant neoplasms.

Cytogenetic and antineoplastic effects by newly synthesised steroidal alkylators in lymphocytic leukaemia P388 cells in vivo.

New compounds with potential antitumour activity were synthesised by combining nitrogen mustard with the steroidal skeleton, in an effort to improve specificity and at the same time reduce systemic toxicity. The steroidal part is aimed to serve as a biological platform enabling the alkylating moiety to approach its site of action by altering its physicochemical properties. The purpose of the present investigation was to evaluate these compounds for anti-neoplastic activity. The compounds tested have as alkylators either para-NN-bis(2-chloroethyl)-aminophenyl-butyrate (CHL) or para-N,N-bis(2-chloroethyl)-aminophenyl-acetate (PHE) esterified with a differently modified steroidal nucleus. The eight newly synthesised compounds were compared on a molar basis with respect to their ability to induce sister chromatid exchanges (SCEs) and to modify proliferation rate indices (PRI) in lymphocytic leukaemia P388 cells in mice in vivo. The life span of BDF1 mice inoculated with P388 leukaemia cells was also estimated (anti-leukaemic activity). The compounds that were effective in inducing cytogenetic effects in lymphocytic leukaemia cells in vivo were also effective in inducing antineoplastic effects in BDF1 mice inoculated with P388 leukaemia cells. These results suggest that the in vivo cytogenetic effects in conjunction with the antineoplastic activity of modified steroidal alkylators depend on the configuration of the whole molecule and on the appropriate combination of the alkylator with the steroidal molecule: a pronounced cytogenetic and anti-neoplastic action was demonstrated by the compounds that contain either PHE or CHL as alkylators and are esterified with either a steroidal nucleus that carries a cholesten group in the 17 position of the D-ring, or with a steroidal nucleus having an exocyclic NHCO-group in the D-ring. In contrast, a ketone group or an NHCO-group in the D-ring inserted endocyclically in the steroidal nucleus esterified with either CHL or PHE failed to induce cytogenetic or anti-neoplastic effects.

In vitro and in vivo cytogenetic effects of recombinant human erythropoietin on the frequency of sister chromatid exchanges alone or in combination with mitomycin C.

BACKGROUND: Erythropoietin (EPO) is a glycoprotein which has a main property, erythropoiesis, but its range of action in the human body is very wide. It has been suggested that EPO acts cytoprotectively for many cell lines against many toxic causes in vitro and in vivo. Our aim was to study the action of EPO on DNA of two cell types, human lymphocytes in vitro and on P388 ascites tumor cells inoculated in BDF1 mice in the presence and absence of the genotoxic agent mitomycin C (MMC). METHOD: The sister chromatid exchange (SCE) assay was used as it is a very sensitive, simple and rapid method for detecting DNA damage. Proliferation rate indices (PRI) and mitotic indices (MI) were also counted. RESULTS: EPO did not alter the SCE level when it acted alone on both cell lines. MMC as a potent genotoxic agent increased SCE levels in vitro and in vivo. EPO used in combination with MMC significantly decreased SCE levels and increased PRI and MI values induced by MMC alone both in vitro and in vivo. CONCLUSIONS: EPO acts protectively against the genotoxic potential of MMC, and this action may have clinical implications.

Preclinical evaluation of amiodarone for the treatment of murine leukemia P388. In vivo and in vitro investigation.

PURPOSE: The purpose of the present study was the investigation of antileukemic effect of amiodarone in leukemia P388 BDF1 bearing mice and its genotoxic and cytostatic effect in cultured normal human lymphocytes. METHODS: Leukemia P388 was used in this study. BDF1 mice were used for chemotherapy evaluation in vivo. The antitumor activity was assessed by the oncostatic parameter T/C, representing the increase of life span of drug-treated animals vs. controls. Lymphocyte cultures were used to study the genotoxic and cytostatic effect in vitro, expressed by enhanced sister chromatid exchange (SCE) and reduced proliferation rate indices (PRIS). RESULTS: Amiodarone was found to exert antileukemic potency against leukemia P388 bearing mice at all three different treatment schedules used, yielding T/C values of 155%, 163% with one cure and 230%. In the in vitro cytogenic experiments, significant increase of SCE rates by amiodarone was observed at 0.2 µM, while at the same concentration significant suppression of PRIS was achieved. CONCLUSION: According to the National Cancer Institute (NCI), a compound is characterized as potential chemotherapeutic deserving further evaluation if it produces T/C values≥125%. On the other hand the SCE assay has predictive value as a clinical assay for drugs exhibiting a strong correlation between cell killing and induction of SCEs. Further studies are warranted to clarify the structure-activity relationship of amiodarone.

Regulation of amyloid A gene expression in cultured cells.

Serum amyloid A (SAA) proteins are secreted by mammalian liver in response to inflammatory stimuli. Both transcriptional and posttranscriptional mechanisms have been shown to regulate the 2,000-fold increase in SAA mRNA after injection of endotoxin into mice. We report here the characterization of a cell line derived from mouse liver (BNL) in which the expression of SAA3 mRNA is regulated. In this model, SAA3 mRNA accumulated in response to conditioned medium from the mouse macrophage P388D1 cell line with kinetics similar to that seen in mouse liver (C. A. Lowell, R. S. Stearman, and J. F. Morrow, J. Biol. Chem. 261:8453-8461, 1986). In in vitro nuclear transcription assays, the SAA3 gene was transcribed equally in induced and uninduced cells. In addition, in steady-state RNA studies treatment with conditioned medium allowed the cells to rapidly accumulate SAA3 mRNA without an apparent change in half-life. These observations suggest that conditioned medium contains a factor(s) that acts directly on hepatocytes to regulate SAA3 mRNA processing.

Physical mapping, amplification, and overexpression of the mouse mdr gene family in multidrug-resistant cells.

The mouse mdr gene family consists of three distinct genes (mdr1, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdr1 (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdr1 (7 of 12) or mdr3 (3 of 12) but not mdr2. mdr1 overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdr1 mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdr1 and mdr3, can independently confer multidrug resistance in the cell lines examined.

Activation of the mouse mdr3 gene by insertion of retroviruses in multidrug-resistant P388 tumor cells.

In multidrug-resistant (MDR) derivatives of the mouse lymphoid tumor P388, the emergence of MDR is associated with overexpression and transcriptional activation of the mdr3 gene, either in the absence of (P388/VCR-10) or concomitant with (P388/ADM-2) gene amplification. In both instances, Northern (RNA) blotting analyses have suggested the presence of altered mdr3 transcripts in these cells, possibly originating from novel transcription initiation sites. The mechanisms underlying mdr3 overexpression in these cells have been investigated. In P388/VCR-10 cells, Southern blotting analyses together with genomic DNA cloning and nucleotide sequencing have demonstrated the presence of an intact mouse mammary tumor virus (MMTV) within the boundaries of intron 1 of mdr3. cDNA cloning and nucleotide sequencing indicated that this integration event results in the synthesis and overexpression of a hybrid MMTV-mdr3 mRNA which initiates within the U3 region of the 5' long terminal repeat (LTR) of the provirus. Consequently, this mRNA lacks the normal exon 1 of mdr3 but contains (i) MMTV LTR-derived sequences at its 5' end, (ii) a novel mdr3 exon, mapping within the boundaries of intron 1 downstream of the MMTV integration site and generated by alternative splicing, and (iii) an otherwise intact 3' portion of mdr3 starting at exon 2. A similar type of analysis of P388/ADM-2 cells revealed that mdr3 overexpression in these cells is associated with the integration of an intracisternal A particle (IAP) within an L1Md repetitive element, immediately upstream of mdr3. The IAP insertion results in the overexpression of hybrid IAP-mdr3 mRNA transcripts that initiate within the 3' LTR of the IAP and which contain IAP LTR-derived sequences at the 5' end spliced 14 nucleotides upstream of the normal exon 1 of mdr3. Taken together, these results indicate that independent retroviral insertions were the initial mutagenic event responsible for mdr3 overexpression and survival during drug selection of these cell lines. Amplification of the rearranged and activated mdr3 gene copy occurred during further selection for high-level drug resistance in P388/ADM-2 cells.

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.

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.

Transcriptional activation of the mouse mdr3 gene coincides with the appearance of novel transcription initiation sites in multidrug-resistant P388 tumor cells.

In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene. In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification. The mechanism underlying mdr3 overexpression in these cells was investigated. Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene. Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1). Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon. In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2. Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Markedly reduced levels of anthracycline-induced DNA strand breaks in resistant P388 leukemia cells and isolated nuclei.

DNA single-strand and double-strand breaks produced by doxorubicin and two anthracycline derivatives (4-demethoxy-daunorubicin and 4'-deoxy-4'-iododoxorubicin) were measured in doxorubicin-sensitive and -resistant P388 leukemia cell lines, using filter elution methods, and compared with cellular drug accumulation to account for major differences in their cytotoxic activities and cross-resistance. The increased cytotoxic potency of the two derivatives reflects at least in part the enhanced drug accumulation by cells that results from their increased lipophilicity. However, the level of protein-linked DNA breaks was not directly related to cellular accumulation of drug analogues. It is possible that enhanced cytotoxicity may also be the consequence of the greatly enhanced ability of analogues to cause DNA strand breaks. The resistant line showed only a modest degree of resistance to both anthracycline derivatives compared with the high degree of resistance to doxorubicin. Although for all the anthracyclines tested drug accumulation was reduced in the resistant line, this did not correlate with the degree of resistance. A differential sensitivity of resistant and parental cell lines to DNA cleavage activity was consistently found for all three drugs tested. However, in contrast to a lack of effect of doxorubicin, the derivatives caused appreciable DNA strand breakage in resistant cells. The enhanced ability of these analogues to break DNA in resistant cells is consistent with the slight cross-resistance with doxorubicin. DNA double-strand breaks produced in isolated nuclei from these cells paralleled the pattern found in whole cells, thus indicating that a nuclear alteration, presumably involving DNA topoisomerases, is associated with anthracycline resistance. Our findings strongly support the hypothesis that anthracycline resistance in these cell variants may be mediated by multiple mechanisms, involving alterations of plasma membrane and changes of nuclear enzymatic activities responsible for DNA strand breaks.

In vivo and in vitro evaluation of the alkylating agent carmethizole.

Carmethizole, a novel bis-carbamate alkylating agent, was evaluated in vitro for potential mechanisms of interaction with DNA and in vivo for spectrum and degree of antitumor activity. In vitro, the concentration of carmethizole required to produce a 50% reduction in clonogenic cell survival was identical in O6-alkylguanine DNA alkyltransferase-positive and -negative human cell lines. The CHO cell line UV4, hypersensitive to mono- and bifunctional alkylating agents, was 37-fold more sensitive to carmethizole than normal cells. The UV5 cell line, which is not hypersensitive to cross-linkers, was 13-fold more sensitive to carmethizole than normal cells. Alkaline elution studies in L1210 cells exposed to carmethizole showed the presence of DNA-protein and DNA-DNA cross-links but not DNA strand breaks. These data suggested that the interaction of carmethizole with DNA produces monoadducts, DNA-protein, and DNA-DNA interstrand cross-links at several sites. In vivo, carmethizole was not cross-resistant with 1,3-bis(2-chloroethyl)-1-nitrosourea or Cytoxan as determined by testing against P388 leukemias resistant to the latter 2 agents. Carmethizole activity was similar to that of melphalan across the murine solid tumor panel, which consisted of B16 melanoma; colon adenocarcinomas 11a, 26, and 36; and the KHT sarcoma. Carmethizole, Cytoxan, and melphalan were all active and had comparable activity against the HCT-8 and MX-1 human tumor xenografts. The in vivo spectrum of activity and efficacy of carmethizole was similar to that of melphalan.

DNA-mediated transfer and cloning of a human multidrug-resistant gene of adriamycin-resistant myelogenous leukemia K562.

We have successfully transferred and cloned a fragment of a human multidrug-resistant gene by using DNA-mediated gene transfer. Macromolecular DNA of human multidrug-resistant K562 cells was transfected to drug-sensitive mouse Ltk- cells to obtain a drug-resistant transfectant with a human resistant gene. Both primary and secondary transfectants showed similar patterns of cross-resistance to Adriamycin and vincristine. The mechanism of drug resistance of the transfectants was attributed to decreased retention of the drug. Three secondary transfectants obtained independently contained common Alu-containing EcoRI fragments 15, 6.5, 3.7, 2.6, and 1.9 kilobases long. The 2.6-kilobase EcoRI fragment was cloned from a lambda phage genomic library made from DNA of a secondary transfectant. The 2.6-kilobase fragment was detected in the primary and secondary transfectants but not in the parental Ltk-, Adriamycin-resistant Ltk-, and Adriamycin-resistant P388 cells. This sequence was found to be amplified in several multidrug-resistant cell lines such as Adriamycin-resistant ovarian carcinoma A2780 and colchicine-resistant KB carcinoma cells. The 2.6-kilobase fragment hybridized with a 4.5-kilobase mRNA which is overexpressed in the Adriamycin-resistant K562 cells and the Adriamycin-resistant A2780 cells but not detected in the parental K562 cells. The gene transferred and cloned in this study seems to be related to the P-glycoprotein gene as judged from the size of mRNA and its overexpression in some of the multidrug-resistant cell lines where P-glycoprotein was found to be highly expressed.

Altered serine/arginine-rich protein phosphorylation and exonic enhancer-dependent splicing in Mammalian cells lacking topoisomerase I.

DNA topoisomerase I (Topo I) specifically phosphorylates arginine-serine-rich (SR proteins) splicing factors and is potentially involved in pre-mRNA-splicing regulation. Using a Topo I-deficient murine B lymphoma-derived subclone (P388-45/C) selected for its resistance to high dosage of the antitumor drug camptothecin, we show that Topo I depletion results in the hypophosphorylation of SR proteins and impairs exonic splicing enhancer (ESE)-dependent but not constitutive splicing. The Affymetrix GeneChip system analysis revealed that several alternatively spliced genes, characterized by small exons and large introns, are down-regulated in Topo I-deficient cells. Given that ectopic expression of green fluorescent protein-Topo I fusion in Topo I-deficient cells restores both wild-type phosphorylation of SR proteins and ESE-dependent splicing, we conclude that Topo I-mediated phosphorylation plays a specific role in ESE-regulated splicing.

Characteristics of P388/VMDRC.04, a simple, sensitive model for studying P-glycoprotein antagonists.

Cross-resistance to chemotherapeutic drugs is a significant problem in the treatment of patients with cancer. The discovery that this phenomenon is associated with the overexpression of a membrane glycoprotein, P-glycoprotein, which acts as a drug efflux pump, has provided a new target for drug development. To develop a model for identifying new compounds which can block the function of P-glycoprotein, we infected P388 mouse leukemic cells with a retrovirus containing a cloned human MDR1 complementary DNA. The new cell line, P388/VMDRC.04, incorporated and overexpressed the human gene as evidenced by Southern blots, increased mRNA and protein synthesis, and recognition by the MRK16 monoclonal antibody. P388/VMDRC.04 was cross-resistant to colchicine, vincristine, and doxorubicin, and the degree of resistance correlated with a reduction in cellular drug accumulation. Unlike many cell lines selected for resistance by growth in increasing concentrations of drug for prolonged periods of time, these cells did not show alternative mechanisms of resistance such as increased synthesis of glutathione or alterations in topoisomerase II. In addition, the sensitivity of P388/VMDRC.04 cells was completely restored by cyclosporin A and trans-flupenthixol. P388/VMDRC.04 cells were subcloned and 10 clones were picked for in vivo evaluation. One subclone grew similarly to parental cells in female BALB/c x DBA/2 F1 mice and showed no responsiveness to therapeutic doses of vincristine or etoposide. The combination of vincristine with cyclosporin A significantly increased the survival of mice inoculated with P388/VMDRC.04 cells. The availability of a cell line that displays the MDR phenotype, overexpresses human P-glycoprotein, but does not contain alterations in at least two well-defined alternative mechanisms of resistance, and that can be grown in simple animal models should facilitate the development of new agents active against this form of chemotherapeutic drug resistance.

Activity of a novel camptothecin analogue, homocamptothecin, in camptothecin-resistant cell lines with topoisomerase I alterations.

Homocamptothecin (hCPT), which differs from camptothecin (CPT) by the presence of an additional methylene group in the E-ring, was evaluated in CPT-resistant cell lines. Topoisomerase I (top1)-deficient leukemia P388/CPT45 cells were highly resistant to hCPT, which demonstrates that top1 is the primary target of hCPT. Three CPT-resistant cell lines with top1 point mutations (Chinese hamster lung fibroblast DC3F/C10, human prostate carcinoma DU-145/RC1, and human leukemia CEM/C2) and their top1 enzymes were cross-resistant to hCPT. The antiproliferative activity of hCPT was greater than that of CPT in both parental and CPT-resistant cell lines, particularly in the prostate cell lines. The top1 cleavage complexes formed in the presence of hCPT appear to be more stable than those induced by CPT. Together, these data indicate that hCPT is a specific top1 inhibitor, which shares a common binding site with CPT in the topl-DNA cleavage complexes. Because of its potency, hCPT might overcome resistance to CPT in some cancer cells.

Thaliblastine, a plant alkaloid, circumvents multidrug resistance by direct binding to P-glycoprotein.

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells. In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [3H]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.

Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506.

Specific phosphorylation of serine- and arginine-rich pre-mRNA splicing factors (SR proteins) is one of the key determinants regulating splicing events. Several kinases involved in SR protein phosphorylation have been identified and characterized, among which human DNA topoisomerase I is known to have DNA-relaxing activity. In this study, we have investigated the mechanism of splicing inhibition by a glycosylated indolocarbazole derivative (NB-506), a potent inhibitor of both kinase and relaxing activities of topoisomerase I. NB-506 completely inhibits the capacity of topoisomerase I to phosphorylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrate activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts competent in splicing but not splicing-deficient cytoplasmic S100 extracts treated with the drug fail to phosphorylate SF2/ASF and to support splicing of pre-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the spliceosome, a dynamic ribonucleoprotein structure where splicing takes place. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spliceosome assembly. Splicing inhibition can be relieved by adding phosphorylated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylating activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant cells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulated hypophosphorylated forms of SR proteins and polyadenylated RNA in the nucleus. In contrast, neither SR protein phosphorylation nor polyadenylated mRNA distribution was affected in P388 CPT5-treated cells. Consistently, NB506 treatment altered the mRNA levels and/or splicing pattern of several tested genes (Bcl-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs targeting topoisomerase I can affect gene expression by modulating pre-mRNA splicing through inhibition of SR proteins phosphorylation.

Investigation of sesquiterpene lactones as protein synthesis inhibitors of P-388 lymphocytic leukemia cells.

The mode of action of helenalin and bis(helenalinyl) malonate as protein synthesis inhibitors of P-388 lymphocytic leukemia cells was investigated. The initial characterizations were carried out in crude lysates of the P-388 cells. In the lysate, there was a 4 min lag after the addition of drug before inhibition of protein synthesis occurred. Both drugs allowed run-off of preformed polysomes, but did significantly inhibit the formation of the 80 S initiation complex suggesting a preferential inhibition of one or more initiation reactions. The effect of these drugs on inhibition of both elongation and initiation reactions was further investigated using more fractionated systems prepared from P-388 cells. Poly(U)-directed polyphenylalanine synthesis was marginally inhibited by both drugs, but the degree of inhibition was not sufficient to explain the inhibition observed in either the lysate or in whole cell preparations of P-388. The formation of the ternary initiation complex was not significantly inhibited by either drug, but the conversion of this complex to the 48 and 80 S initiation complexes was inhibited. The inhibition of 48 S initiation complex formation by both drugs was sufficient to explain their inhibition of protein synthesis in whole cells.

Cytogenetic analysis of the bipotential murine pre-B cell lymphoma, P388, and its derivative macrophage-like tumor, P388D1, using SKY and CGH.

Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) were used to elucidate the divergent cytogenetic make-up of the prototypical bilineage lymphoblastic pre-B lymphoma, P388, and its progenitor macrophage-like tumor, P388D1. P388 was found to be diploid and genomically stable. P388D1 was triploid, highly unstable and characterized by numerous marker chromosomes (Chrs) and composite rearrangements. The karyotype of P388D1 was so complex that its clonal relatedness to P388 would have remained questionable without confirmation by molecular analysis of the clonotypic immunoglobulin heavy-chain and light-chain gene recombinations that coexisted in both tumors. The intrinsic instability of the P388D1 genome was indicated by the observation that only four out of 42 aberrations uncovered by SKY (in a total of 27 metaphases) occurred consistently (100% incidence), whereas 27 changes occurred non-randomly (27 to 96% incidence) and 11 alterations randomly (4 to 11% incidence). Persistent cytogenetic instability was also observed in P388 'macrophages' after phorbol ester- and ionomycin-induced conversion in vitro of P388 lymphoma cells. The 'cytogenetic noise' in these cells was manifested by a multiplicity of sporadic chromosomal aberrations; ie 25 distinct changes were identified by SKY in 40 metaphases. The results in P388D1 and P388 'macrophages' were interpreted to indicate that the myeloid differentiation program in the bipotential pre-B cell lymphoma P388 is invariably characterized by karyotypic instability. The study presented here demonstrates the power of the combined SKY and CGH approach to resolve complicated karyotypes of important and widely used mouse tumors.