Topoisomerase II activity involved in cleaving DNA into topological domains is altered in a multiple drug-resistant Chinese hamster ovary cell line.

Drug resistance to inhibitors of DNA topoisomerase II can result from qualitative or quantitative alterations in the target enzyme, topoisomerase II, or from perturbations in drug transport that may or may not involve P-glycoprotein. In the present study, a drug-resistant Chinese hamster ovary cell line, SMR16, was selected in the presence of an epipodophyllotoxin (VP-16) and was found to be cross-resistant to all classes of topoisomerase II inhibitors (3-35-fold). The 3-fold level of resistance of these cells to vincristine is likely due to diminished uptake of this drug, and this is not mediated by overexpression of P-glycoprotein. No alteration in transport of VP-16 was observed. Immunoblotting with several polyclonal anti-topoisomerase II antibodies demonstrated that the resistant cells contain approximately two-thirds of the parental enzyme amount. The topoisomerase II catalytic activity present in 0.35 M NaCl nuclear extracts paralleled this decrease. VP-16- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA damage, mediated by topoisomerase II, was found to be decreased 10-12-fold in both intact SMR16 cells and nuclei isolated from these cells, when measured by alkaline filter elution. However, the VP-16-induced DNA cleavage activity present in 0.35 M NaCl nuclear extracts of the resistant cells was attenuated only 2-fold, relative to wild-type cells. Homogeneous preparations of the enzyme obtained from resistant cells demonstrated the same cleavage and catalytic activity as purified wild-type topoisomerase II. Analysis by pulse-field gel electrophoresis of the DNA isolated from VM-26- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-treated sensitive and resistant cells demonstrated significantly less conversion of SMR16 chromosomal DNA into 50-150-kilobase DNA fragments. Chinese hamster ovary SMR16 cells are apparently resistant to topoisomerase II poisons because the topoisomerase II that defines the DNA topological domains is either decreased in amount or insensitive to drug action.

Heterogeneity of hairy cell tartrate-resistant acid phosphatase.

The human nonerythrocytic acid phosphatases (AcP) are composed of seven distinct activity bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) when stained using either 1-naphthyl phosphate or naphthol ASBI phosphate as substrate. They are numbered 0, 1, 2, 3, 3b, 4, and 5 according to their increasing mobility toward the cathode in acidic conditions. Of these, only the most cationic "band 5" is tartrate resistant (TRAcP). When naphthol ASBI phosphate is used as substrate, AcP activity can also be stained in situ. In the presence of tartrate, activity remains strong in the hairy cells (HC) of hairy cell leukemia (HCL). Thus, the TRAcP stain has remained a reliable marker for HC. To investigate the function of TRAcP in HC, we purified two isoforms of TRAcP from HCL spleen tissue and found them to have similar substrate specificities and inhibitor sensitivities. In this report, we describe in detail the methods for TRAcP purification and compare some of the structural properties of the two isoforms to reinforce the concept that human TRAcP is a heterogeneous group of related enzymes. Band 5 represented only 15-20% of the total TRAcP extracted from HCL spleen. The remaining 80% of TRAcP hydrolyzed p-nitrophenyl phosphate but not naphthol ASBI phosphate and was not detectable in acidic, nondenaturing PAGE gels. Band 5 was solubilized from tissue using 500 mmol/L NaCl after previous extraction with 0.5% (v/v) NP-40 removed most other AcP and TRAcP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuated topoisomerase II content directly correlates with a low level of drug resistance in a Chinese hamster ovary cell line.

A new multiple drug-resistant Chinese hamster ovary cell line, CHO-SMR5, has been isolated which demonstrates a direct correlation between reduced cellular topoisomerase II activity (5-fold reduction) and a low level of resistance (3- to 7-fold) to topoisomerase II inhibitors. This cell line, initially selected for resistance to 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin, exhibits cross-resistance to other topoisomerase II inhibitors including 4'-(9-acridinylamino)methanesulfon-m-anisidide, doxorubicin, and mitoxantrone. The resistant cells show a 4.5-fold decrease in topoisomerase II by immunoblotting when compared to wild-type cells. Drug uptake studies reveal equivalent equilibrium intracellular concentrations of [3H]9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethyepipodophyll otoxin in the resistant and parental cells. The catalytic activity of topoisomerase II (decatenation of kinetoplast DNA) is 5-fold less in the drug-resistant cell line relative to wild-type Chinese hamster ovary cells. Drug-induced DNA damage, measured as either formation of DNA double-strand breaks or covalent DNA-enzyme complexes, is 4-fold less in the resistant cell line. Finally, Northern blot analysis demonstrates a 5-fold reduction in topoisomerase II mRNA isolated from log phase CHO-SMR5 cells. These findings suggest that a reduced level of topoisomerase II is likely to be the sole mechanism of drug resistance in this novel cell line.

Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line.

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes bahamensis: its invasion of south Florida and association with Aedes aegypti.

The exotic mosquito, Aedes bahamensis, is now well-established in south Florida, where it is widely distributed throughout Dade and southern Broward Counties in both urban and rural areas east of the Everglades. When discarded automobile tires were sampled in areas near human habitation, larvae and pupae of Ae. bahamensis were frequently found in association with immature Ae. aegypti. Elsewhere, however, Ae. bahamensis generally occurred in the absence of Ae. aegypti. The persistence of Ae. bahamensis populations at specific sites was documented in egg collections from ovitraps and in larval samples from the water retained in discarded tires.

Inhibition of topoisomerases by fredericamycin A.

Fredericamycin is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro. Because of its unique structure and the absence of a clearly defined mechanism of action, we examined the effects of this compound on L1210 cells in culture as well as on several enzymes that bind to DNA. Fredericamycin exhibits an IC50 of 4.4 microM toward L1210 cells, and its cytotoxicity is a function of the time of exposure as well as drug dose. No DNA breakage was observed in L1210 cells or isolated nuclei following exposure to highly lethal concentrations of fredericamycin. As a first step toward understanding its mechanism of action, we examined the effect of fredericamycin on several enzymes involved in DNA metabolism. The catalytic activity of both DNA topoisomerases I and II were totally inhibited by fredericamycin concentrations of 4.4 and 7.4 microM, respectively. Fredericamycin blocked etoposide-stimulated DNA cleavage by topoisomerase II both in vitro and in isolated nuclei. In addition, the drug inhibits DNA polymerase a in vitro, exhibiting an IC50 of 93 microM. These diverse actions of fredericamycin do not enable us to draw conclusions regarding its mechanism of antitumor effect but clearly identify it as a compound of pharmacologic interest.

Proliferation-dependent topoisomerase II content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells.

We have shown previously that quiescent Chinese hamster ovary (CHO) cells are less sensitive than log phase CHO cells to the cytotoxic and DNA cleavage effects of etoposide, a drug which appears to act via DNA topoisomerase II. This loss of sensitivity was associated with a decrease in topoisomerase enzyme activity in nuclear extracts of the quiescent cells. We have now extended our observations by examining the basis for the reduction in enzyme activity during quiescence. DNA topoisomerase II content, as assayed by immunoblotting with a polyclonal rabbit anti-topoisomerase II antiserum, was virtually absent in nuclear extracts of quiescent CHO cells in contrast to logarithmically growing cells. This suggests that the previously demonstrated loss of enzyme activity in CHO cells is a function of reduction in content rather than posttranslational modifications of the enzyme. Quiescent human lymphoblastic CCRF cells also exhibited reduced topoisomerase II content compared to actively proliferating cultures, but the difference was less than that observed in CHO cells. In contrast, log and plateau phase cultures of mouse leukemia L1210 cells exhibited similar topoisomerase II content. Reduction in enzyme content correlated with the ability of these cell lines to accumulate during quiescence with a G0-G1 content of DNA. Sensitivity to the DNA cleavage effects of etoposide in dividing and nondividing cells correlated well with enzyme content. As has been observed with CHO cells, both CCRF and L1210 cells in plateau phase were more resistant to the cytotoxic effects of etoposide than those actively dividing. The result with L1210 cells was surprising, however, in light of the equivalent DNA damage observed under the two growth conditions. Our data indicate that topoisomerase II enzyme content is proliferation dependent in some but not all cells and suggest that while enzyme content may be important in drug resistance in some cell types, other factors can decrease the sensitivity of the cell to cleavable complex formation as well.