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PubMed2000

Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors.

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.

Animals↗

PubMed2000

Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis.

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.

Amino Acid Sequence↗

PubMed1995

Biochemical modulation of cytotoxic drugs by cytokines: molecular mechanisms in experimental oncology.

The combination of cytokines and cytotoxic drugs offers a new approach to increase the therapeutic index in the treatment of neoplastic diseases. There is no consensus on optimal strategies for combining these agents so far. The molecular mechanisms underlying the interaction, however, should be defined in order to design clinical trials based on preclinical rationales. The broad spectrum of cytotoxic drugs whose activity can be enhanced by cytokines argues for multiple levels of drug interaction in vitro: alteration in the cellular drug uptake, modulation of drug target enzymes, and changes in metabolism or disposition of a drug. In vivo interaction between cytokines and cytotoxic agents involves an additional layer of complexity because of the effects of cytokines on the host immune system and on drug-metabolizing enzymes. A major mechanism involved in the synergistic interaction of interferon (IFN) and 5-fluorouracil (5-FU) seems to be the increase of active 5-FU metabolites by IFN. Moreover, IFN can reverse resistance against 5-FU by inhibiting the overexpression of thymidylate synthase. The absence of cytokinetic effects of IFN and FU argues against the recruitment of Gs cells into the cell cycle. Topoisomerase has emerged as a critical intracellular target of cytotoxic drugs. There is convincing evidence that the synergy between tumor necrosis factor (TNF) and topoisomerase-targeted intercalative (Adriamycin, doxorubicin hydrochloride; m-AMSA, amsacrine; mitoxantrone) and nonintercalative (VM-16, etoposide; VM-26, teniposide) drugs is related to a rapid increase in specific activity of topoisomerase I and II, resulting in enhanced DNA strand breaks and cleavage complex. Furthermore, sensitivity to topoisomerase II targeted drugs can be enhanced by granulocyte colony-stimulating factor (G-CSF) through elevated enzyme activity in tumor cell response to G-CSF. The synergistic interaction between cytokines and cytotoxic agents seems to be sequence dependent. It has recently been demonstrated that newly synthesized metal compounds and IFN are synergistic only after preincubation with cytokines. Cytokines can modulate expression of adhesion receptors on tumor cell lines, thereby influencing their metastatic potential. A considerable number of phase II trials with combination of cytokines and cytotoxic drugs based on these mechanisms have demonstrated promising response rates and tolerable toxicity. Phase III trials are currently in progress to identify enhanced activity combining cytokines and cytotoxic drugs in the treatment of malignancies.

Animals↗

PubMed1988

Clinical trial of etoposide (VP-16) in children with recurrent malignant solid tumors. A phase II study from the Pediatric Oncology Group.

Etoposide (VP-16), 150 mg/M2, given intravenously daily for 3 days every 3 weeks resulted in 3 complete responses and 6 partial responses in 154 patients with a spectrum of recurrent malignant solid tumors. There was evidence of disease control in an additional 37 patients (27 mixed responses and 10 stable disease). These responses occurred primarily in patients with Ewing's sarcoma, Hodgkin's disease, neuroblastoma and rhabdomyosarcoma. Most of the patients had every extensive prior therapy; however prior therapy with teniposide (VM-26), the congener of VP-16, did not seem to preclude responses to the latter drug. Myelosuppression was the principal form of toxicity. Neutropenia characterized by absolute neutrophil counts of 0.5 to 0.9 x 10(9)/L occurred in one-half of the patients, and thrombopenia with platelet counts of less than 25 to 49 x 10(9)/L in one-fourth. These results demonstrate a favorable therapeutic index for VP-16 in several recurrent childhood solid tumors, supporting its use as a component of primary therapy for these diseases.

Adolescent↗

PubMed1987

Etoposide-induced DNA cleavage in human leukemia cells.

The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents such as etoposide, teniposide, and a variety of intercalating agents. These compounds cause the enzyme to cleave DNA, forming a DNA-protein complex that may be a key step leading to cell death. It is apparently unique as a chemotherapy target, since drug potency diminishes with decreasing enzyme activity. It was thus of interest to examine the topoisomerase content and drug-induced DNA cleavage in freshly obtained human leukemia cells and to compare the obtained data with the results of similar studies performed in well-characterized human leukemia cell lines. The human T-lymphoblast line, CCRF-CEM, was more than 100-fold more sensitive to the DNA-cleavage effect of etoposide than the cells of the 13 leukemic patients examined. One of the leukemia lines (HL-60) and a lymphoblastoid line (RPMI-7666) were somewhat less sensitive than cells of the CCRF-CEM cells, but were still 10-fold more sensitive than the patients studied. The relative insensitivity of the freshly obtained cells could not be accounted for by differences with respect to drug uptake but were associated with markedly reduced topoisomerase-II content as assayed by immunoblotting using a mouse polyclonal serum against topoisomerase II. Heterogeneity was observed in the sensitivities of patients' cells with respect to both drug-induced DNA cleavage and enzyme content. The observed differences between cultured cell lines and patients' cells may have been related to their proliferative status. Etoposide potency in normal resting lymphocytes resembles that observed in circulating leukemia cells. However, following mitogenesis with phytohemagglutinin and interleukin-2, proliferating lymphocytes become as sensitive to etoposide as cultured cell lines with regard to DNA cleavage. This effect was accompanied by an increase in topoisomerase-II content. Our data thus support the hypothesis that topoisomerase-II content may be an important determinant of cell sensitivity to certain classes of chemotherapy agents. Efforts to stimulate topoisomerase-II content may improve the therapeutic efficacy of these drugs.

Cell Division↗

PubMed1983

Optimum scheduling during combination chemotherapy of murine leukemia. Additional examples of schedule-dependent synergism between S-phase-specific antimetabolites and agents inducing mitotic or pre-mitotic (G2) arrest.

In an extension of our prior studies with methotrexate and vinca alkaloids, three additional drug combinations incorporating an S-phase-specific antimetabolite and an agent inducing blockade at mitosis or G2 were found to exhibit potent schedule-dependent synergism against the L1210 leukemia. Combinations employed include cytosine arabinoside with vindesine and methotrexate with teniposide (VM-26) or the -deaza-pteridine derivative, NSC 181,928. Synergism was observed following sequential administration (antimetabolite given 24 h before the second agent), but effects following simultaneous administration or sequential administration in the reverse order (antimetabolite given 24 h after the second agent) were only additive.

Animals↗

PubMed1988

Study of histamine release induced by acute administration of antitumor agents in dogs.

The effects of eight antitumoral drugs known to cause anaphylactoid side effects in clinical use were studied in dogs. Blood pressure, heart rate, and blood and plasma histamine levels were monitored. L-asparaginase, methotrexate, 5-fluorouracil, bleomycin, and cisplatin had no effect on these parameters. Doxorubicin, Vehem (teniposide), and Vepeside (etoposide) induced hypotension, tachycardia, and a rise in histamine levels. In the cases of Vehem and Vepeside, the excipient (respectively, cremophor EL and tween 80) induced the same effects. These agents, like elliptinium, which had been previously studied, induce nonspecific histamine release--unlike the other drugs studied. The mechanism of clinically observed anaphylactoid side effects is discussed in the light of these findings.

Anaphylaxis↗

PubMed1980

Time-dependent changes of plasma ribonuclease activity in female Wistar rats under combination therapy of ovarian carcinoma.

Determinations of plasma ribonuclease were recommended in 1977 by Sheid et al. for laboratory diagnosis of ovarian carcinoma. We investigated the turnover of transfer ribonucleic acid (tRNA) as the substrate of the enzyme in healthy female inbred rats under the multistep-massive dose therapy of ovarian carcinoma usual at the Department of Gynecology, University of Giessen. Whereas mitopodozide, percutaneous irradiation of the abdomen and teniposide did not have any detectable influence on the enzyme activity, the enzyme was strongly suppressed by the other therapy steps: minima of tRNA turnover were observed on the 2nd day with adriamycine, on the 4th day with methotrexate and methotrexate/folinic acid, on the 6th day with cyclophosphamide and fractionated percutaneous irradiation with cyclophosphamide medication in the intervening periods. According to the results, a plasma ribonuclease determination to check the result of therapy should only be performed at an interval of 2 weeks after each therapy step.

Animals↗

PubMed1985

Time-schedule dependency of the inhibiting activity of various anticancer drugs in the clonogenic assay.

To analyze the discrepancy between the in vitro response in the clonogenic assay and the clinical response, the time-schedule dependencies of various anticancer drugs were determined by comparing the inhibiting effect against colony formation by PC-7 cells treated with the drugs for 1 h with that of those treated for 24 h. According to their schedule dependency the drugs can be divided into a schedule-dependent drug group (5-fluorouracil, methotrexate, bleomycin, pepleomycin, etoposide, cisplatin, teniposide, vindesine, and vinblastine) and a non-schedule-dependent drug group (adriamycin, actinomycin D, ranomustine, mitomycin C, aclacinomycin, daunomycin, nimustine, melphalan, and KW 2083). In the clonogenic assay, the 1-h exposure schedule is appropriate for predicting clinical response for the non-schedule-dependent drugs. However, the effect of the schedule-dependent drugs was underestimated in the same conditions. Therefore, it is necessary to test these drugs in the assay by 24-h exposure for a more accurate assessment of their antitumor activity.

Adenocarcinoma↗

PubMed1992

Non-immunological release of histamine from rat mast cells elicited by antineoplastic agents.

We studied the histamine-releasing activity of several antineoplastic drugs on rat pleural and peritoneal mast cells. The drugs tested included the nitrogen mustards cyclophosphamide and ifosfamide, the nitrosourea carmustine, the triazene dacarbazine, the folic acid analogue methotrexate, the pyrimidine analogue cytarabine and fluorouracil, the vinca alkaloids vinblastine, vincristine and Vinorelbine, the epipodophyllotoxins etoposide and teniposide, and the enzyme L-asparaginase. Methotrexate, carmustine, fluorouracil, vinblastine and vincristine failed to elicit histamine release on rat mast cells. All of the other drugs evoked histamine release in both the presence and the absence of extracellular calcium, but ifosfamide, cytarabine and asparaginase induced a much lower release in the absence of this cation. The response elicited by cytarabine and etoposide was much higher in pleural than in peritoneal mast cells. These results indicate that some antineoplastic drugs may directly activate the release of histamine, which could contribute to some of their secondary effects.

Animals↗

PubMed1994

Characterization of an etoposide-resistant human ovarian cancer cell line.

Etoposide (VP-16) is one of the most important anticancer agents available and is used in many chemotherapeutic regimens. To characterize resistance to this drug, we established a VP-16-resistant human ovarian cancer cell line, SKOV3/VP, by continuous stepwise exposure of SKOV3 cells to VP-16. The degree of resistance to VP-16 of SKOV3/VP was about 25 times that of the parent cell line (SKOV3), and SKOV3/VP showed cross-resistance to teniposide, adriamycin, CPT-11, and vincristine. The accumulation of [3H]-VP-16 observed in SKOV3/VP cells was about half that seen in SKOV3 cells, and the accumulation of Adriamycin by this resistant cell line was also lower than that of its parent. Overexpression of neither the multidrug resistance gene mdr-1, the multidrug-resistance-associated protein (mrp) gene, nor P-glycoprotein was detected using reverse transcriptase-polymerase chain reaction analysis and flow cytometry with MRK-16, a monoclonal antibody against P-glycoprotein. The topoisomerase II activity of nuclear extracts from SKOV3/VP cells was lower than that from the parental cells, as was the amount of DNA topoisomerase II, demonstrated by immunoblotting. These results suggest that the mechanism responsible for the multidrug resistance of this cell line may be attributable to changes on its DNA topoisomerase II and to its reduced accumulation of the drugs as compared with the parental line SKOV3.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

In vivo inhibition of etoposide-mediated apoptosis, toxicity, and antitumor effect by the topoisomerase II-uncoupling anthracycline aclarubicin.

A number of clinically important drugs such as the epipodophyllotoxins etoposide (VP-16) and teniposide (VM-26), the anthracycline daunorubicin and doxorubicin (Adriamycin), and the aminoacridine amsacrine exert their cytotoxic action by stabilizing the cleavable complex formed between DNA and the nuclear enzyme topoisomerase II. We have previously demonstrated in several in vitro assays that the anthracycline aclarubicin (aclacinomycin A) inhibits cleavable-complex formation and thus antagonizes the action of drugs such as VP-16 and daunorubicin. The present study was performed to validate these in vitro data in an in vivo model. At nontoxic doses of 6 and 9 mg/kg, aclarubicin yielded a marked increase in the survival of non-tumor-bearing mice given high doses of VP-16 (80-90 mg/kg) in six separate experiments. In therapy experiments on mice inoculated with Ehrlich ascites tumor cells, aclarubicin given at 6 mg/kg roughly halved the increase in median life span induced by VP-16 at doses ranging from 22 to 33 mg/kg. An attempt to determine a more favorable combination of VP-16 and aclarubicin by increasing VP-16 doses failed, as the two drugs were always less effective than VP-16 alone. The way in which VP-16-induced DNA strand breaks lead to cell death remains unknown. However, VP-16 has been reported to cause apoptosis (programmed cell death) in several cell lines. To ascertain whether the protection given by aclarubicin could have a disruptive effect on the apoptotic process, we used the small intestine as an in vivo model. Whereas VP-16-induced apoptosis in crypt stem cells was detectable at a dose as low as 1.25 mg/kg, aclarubicin given at up to 20 mg/kg did not cause apoptosis. Indeed, aclarubicin caused a statistically significant reduction in the number of cells rendered apoptotic by VP-16. The present study thus confirms the previous in vitro experiments and indicates the value of including an in vivo model in a preclinical evaluation of drug combinations.

Aclarubicin↗

PubMed1995

Experimental solid tumour activity of N-[2-(dimethylamino)ethyl]-acridine-4-carboxamide.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA), a DNA intercalator that exerts its antitumour action through the enzyme topoisomerase II, has previously been shown to be curative against the transplantable Lewis lung adenocarcinoma growing as lung tumour nodules in mice. On the basis of this finding as well as its high in vitro activity against multidrug-resistant cell lines, DACA has been chosen for clinical trial under the auspices of the Cancer Research Campaign, United Kingdom. In the present study the activity of DACA was assessed against advanced (5-mm diameter) s.c. colon 38 adenocarcinomas in BDF1 mice using tumour-growth delay as an end point. Its activity was found to be related positively to the total dose given and negatively to the total duration of the dose schedule. Adoption of a split-dose i.p. administration schedule or slow i.v. infusion allowed the administration of large doses without toxicity. The activity of DACA was comparable with that of 5-fluorouracil and superior to that of doxorubicin, cyclophosphamide and the experimental amsacrine analogue CI-921. Mitoxantrone, amsacrine, etoposide, teniposide and daunorubicin showed minimal activity. DACA also demonstrated significant activity against the NZM3 melanoma human cell line growing as a xenograft in athymic mice.

Acridines↗

PubMed1994

An open, randomized study to compare the efficacy and tolerability of tropisetron with that of a metoclopramide-containing antiemetic cocktail in the prevention of cisplatin-induced emesis.

In a prospective randomized study comprising 66 women treated for gynecologic malignancies with cisplatin-containing chemotherapy, the new 5-hydroxytryptamine3 (5-HT3) receptor antagonist tropisetron (Navoban, Sandoz Pharma Ltd.) was compared with a metoclopramide cocktail for the prevention of nausea and emesis. All patients were chemotherapy-naive. Two consecutive courses (including the 1st week posttherapy) were studied. The cisplatin doses were in the range of 50-75 mg/m2, and the regimens also contained doxorubicin, teniposide, etoposide, vincristine, and bleomycin. Complete protection against nausea during the first 24 h (course 1) was achieved in 76% of the tropisetron group and in 85% of the metoclopramide group. Emesis was prevented in 82% of the patients in both groups. During the whole 6-day period, full emetic protection was achieved in 30% and 18% of the patients in the two groups. On days 3-4 of course 1, tropisetron was superior to metoclopramide. The overall tolerability of the tropisetron was excellent or good in 94% of patients, a rate higher than that observed for the metoclopramide regimen (75%). The most common side effects for the latter regimen were sedation (82%) and extrapyramidal reactions (21%). The only significant adverse event recorded after treatment with tropisetron was headache of slight or moderate grade.

Adult↗

PubMed1995

High frequency of mutagen-induced chromatid exchanges at interstitial telomere-like DNA sequence blocks of Chinese hamster cells.

Interstitial telomere-like DNA sequence arrays of Chinese hamster Don cells were delineated by fluorescence in situ hybridization in quadriradial and triradial chromosome configurations induced by X-rays, mitomycin C and teniposide (VM-26). Around 40% of the scored exchanges involved a telomeric-like block of sequences at a rearrangement site. This frequency was independent of the DNA-damaging agent, and the result suggests a general recombinogenic capacity of interstitial telomere-like DNA sequence repeats that, at least in the case of the agents employed, seems not to be related to the initial mechanism of DNA damage.

Animals↗

PubMed1994

Postincubation with aclarubicin reverses topoisomerase II mediated DNA cleavage, strand breaks, and cytotoxicity induced by VP-16.

In previous studies, we found that VP-16 (etoposide) induced cytotoxicity and protein-concealed strand break formation was prevented in a small cell lung cancer (SCLC) cell line, when the cells were incubated with aclarubicin prior to treatment with VP-16. In the present work, we studied the effect of adding aclarubicin to the cell suspension after VP-16. In a clonogenic assay, we found that the cytotoxicity induced by VP-16 in SCLC cells was inhibited when cells were postincubated with aclarubicin. The addition of aclarubicin at any time in relation to VP-16 was able to stop further cytotoxicity induced by the topoisomerase II (topo-II) targeting drug. Aclarubicin was also found to antagonize the cytotoxicity induced by VM-26 (teniposide), and m-AMSA. With the alkaline elution technique we found that postincubating the cells with aclarubicin inhibited VP-16-induced DNA strand break formation. In an in vitro system with purified topo-II and naked DNA we likewise found, that postincubation with aclarubicin prevented VP-16 induced cleavage. In the same in vitro system, also baseline cleavage induced by topo-II was inhibited when aclarubicin was present. Importantly, aclarubicin exerted the antagonism to topo-II targeting drugs both when administered prior to and after the topo-II targeting agents. Thus, our data suggest that sequential rather than simultaneous administration of aclarubicin and topo-II targeting agents may be superior with respect to net-cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aclarubicin↗

PubMed1994

Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell lines and certain drug resistant sublines in vitro.

The in vitro cytotoxic effects of docetaxel (Taxotere; RP56976, NSC688503) proved both time and concentration dependent. Amongst thirteen human cell lines from various tumor types, exposure to increasing concentrations of docetaxel over 24 hrs resulted in a plateau-shaped dose response curve, suggesting that increased cell kill becomes more dependent on increased exposure duration than on concentration. IC50 concentrations (reducing survival by 50%) ranged from 0.13-3.3 ng/ml, with three neuroblastoma lines proving most sensitive and three breast and two colon carcinoma lines showing least sensitivity. There was significant cross-resistance to docetaxel in the classic multidrug resistant (MDR) Chinese hamster ovarian (CHO) CHRC5 line and the human lymphoblastoid CCRF-CEMVLB1000 line, as well as in two vincristine (VCR)-selected MDR MCF-7 sublines. All four of these MDR sublines overexpress P-glycoprotein (Pgp), as did a 6-fold docetaxel-selected resistant CHO subline. As an apparent corollary, in two human teratoma lines selected for etoposide resistance and showing some cross-resistance to VCR and in two CHO sublines expressing low levels of VCR resistance, yet all proving Pgp positive, no docetaxel cross-resistance was identified. Verapamil modulated docetaxel resistance only in sublines expressing resistance to the drug and overexpressing Pgp. Four other human tumor sublines selected for resistance to 5-fluorouracil, cisplatin or teniposide, showed a lack of cross-resistance to docetaxel. Furthermore, cross-resistance to docetaxel was not apparant in four epipodophyllotoxin-selected resistant sublines with alterations in topoisomerase II, indicating its effectiveness against tumor cells expressing the topoisomerase II-related MDR phenotype. Our observation that docetaxel cross-resistance was not automatically expressed by classic MDR tumour cells appears of interest and of potential clinical relevance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation.

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.

Animals↗