Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin.

Camptothecin, a plant alkaloid with antitumor activity, has been shown to be a potent inhibitor of nucleic acid synthesis and a strong inducer of DNA strand breaks in mammalian cells. Previous studies have shown that camptothecin inhibits purified mammalian DNA topoisomerase I by trapping a reversible enzyme-DNA "cleavable complex" (Hsiang et al., J. Biol. Chem., 260: 14873-14878, 1985). Our present studies, using L1210 cells and SV40-infected monkey cells, have shown that camptothecin-induced strand breaks are protein linked. The linked protein is most likely DNA topoisomerase I as revealed by immunoblot analysis, using antibodies against purified mammalian DNA topoisomerase I. Brief heating of camptothecin-treated cells to 65 degrees C resulted in a rapid reduction of the number of protein-linked DNA breaks. Reversal of the camptothecin-induced topoisomerase I-DNA complex by heat was also observed in an in vitro system by using purified mammalian DNA topoisomerase I. Our results suggest that camptothecin interferes with DNA topoisomerase I both in cultured mammalian cells and in the purified system by trapping a reversible enzyme-DNA cleavable complex.

Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin.

Camptothecin, which induces an unusual type of DNA damage by trapping cellular topoisomerase I on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes, inhibits DNA synthesis and specifically kills S-phase cells. Cotreatment of L1210 cells with aphidicolin, which is an inhibitor of replicative DNA polymerases, completely abolished camptothecin cytotoxicity, suggesting the involvement of DNA replication in camptothecin cytotoxicity. In order to study the role of DNA replication in drug action, a cell-free SV40 DNA replication system was used in the present study. Camptothecin inhibited SV40 DNA replication in this cell-free system only in the presence of topoisomerase I. Addition of excess purified calf thymus DNA topoisomerase I to this extract system in the presence of camptothecin resulted in severe inhibition of SV40 DNA replication and the accumulation of linearized replication products, which contained covalently bound DNA topoisomerase I. We propose that the collision between moving replication forks and camptothecin-stabilized topoisomerase I-DNA cleavable complexes results in fork arrest and possibly fork breakage, which are lethal to proliferating cells.

Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells.

The intracellular level of DNA topoisomerase II appears to be reversibly regulated by serum concentration in cultured primary human skin fibroblasts (HSF). Upon serum starvation, the intracellular level of topoisomerase II in HSF, as monitored by immunoblotting with antitopoisomerase II antibodies, gradually decreased to a nondetectable level (less than 10(4) copies/cell) over a period of 72 h. Addition of 10% serum to the starved cells led to a gradual increase of the intracellular topoisomerase II to the original level (approximately 10(6) copies/cell) over a period of 24 h. The intracellular DNA topoisomerase II level in HSF is also sensitive to cell density; minimally a 7-fold decrease was observed when HSF were grown to saturation density in a constant serum concentration. Similarly, the intracellular levels of DNA topoisomerase II in other "nontransformed" cells such as mouse NIH 3T3 and 3T6 cells are also sensitive to both the serum concentration and the cell density. In contrast, topoisomerase II levels in transformed cells such as HeLa cells, L1210 cells, and SV40 T-antigen-transformed COS-1 cells are maintained at high levels (approximately 10(6) copies/cell) and are much less sensitive to growth conditions. The topoisomerase II level in HeLa cells synchronized by a double thymidine block remained relatively constant (less than 2-fold difference) throughout the late G1, S, G2, and M phases of the cell cycle. Our results suggest that the level of DNA topoisomerase II is primarily regulated in the G0-G1 phase of the cell cycle and is elevated to a high level (approximately 10(6) copies/cell) in proliferating cells. In contrast, the intracellular levels of DNA topoisomerase I in these cells were largely unaffected by these growth conditions either in HSF or in HeLa cells.

DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II.

Antitumor drugs from many chemical classes have been shown to induce protein-linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane-sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug-induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines.

Lack of drug-induced DNA cross-links in chlorambucil-resistant Chinese hamster ovary cells.

Chlorambucil (CLB) is an alkylating agent commonly used in the treatment of several neoplastic disorders. The mechanisms underlying resistance to this drug are not fully defined. We used the DNA alkaline elution technique to study cross-link formation in the wild type (K1) and a CLB-resistant (ChlR) Chinese hamster ovary cell line. [14C]CLB was used to measure drug uptake. The CLB-resistant cells were found to have negligible DNA cross-link formation compared to K1 cells at all time points tested. There was a correlation between the resistance to CLB and the decreased ability of resistant cells to form DNA cross-links. Results of drug uptake experiments excluded altered CLB accumulation as the basis for these findings. Assays of O6-alkylguanine transferase and topoisomerase. II provide evidence against a role of these enzymes in CLB resistance. These studies suggest that the mechanism of CLB cytotoxicity involves the formation of DNA cross-links. Reduced cross-link formation may confer resistance to CLB.

Growth-related elevations of DNA topoisomerase II levels found in Dunning R3327 rat prostatic adenocarcinomas.

The relationship between DNA topoisomerase II expression and mammalian cell proliferation has been evaluated by determining enzyme levels in normal and neoplastic rat prostate tissues. By activity assay and by immunoblot analysis using anti-topoisomerase II antiserum, topoisomerase II levels were found to be elevated in both the Dunning R3327-H and the Dunning R3327-G rat prostatic adenocarcinomas over levels assayed in the normal rat dorsal prostate. Immunohistochemical studies using the antitopoisomerase II antiserum revealed that a greater fraction of nuclei contained detectable levels of topoisomerase II in tissue sections prepared from each of the Dunning tumors than in rat dorsal prostate tissue sections. The Dunning R3327-H and R3327-G tumors grow at different rates in vivo (J. T. Isaacs and D. S. Coffey, Clin. Oncol., 2: 479-498, 1983). When measured topoisomerase II levels were compared to known growth parameters for each of the tissues studied, topoisomerase II expression was found to be correlated with tissue growth rate.

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II.

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues.

20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.

Comparative study of home blood glucose monitoring devices: Visidex, Chemstrip bG, Glucometer, and Accu-Chek bG.

Visidex (Ames Company, Elkhart, Indiana) and Accu-Chek bG (Bio-Dynamics, Boehringer Mannheim, Indianapolis, Indiana), two new devices for determining blood glucose at home, were compared with two older devices, Chemstrip bG (Bio-Dynamics) and Glucometer (Ames Company), and the standard laboratory glucose-oxidase method (Beckman autoanalyzer, Beckman Instruments, Fullerton, California). Laboratory serum glucose values up to 400 mg/dl had excellent overall correlation with Accu-Chek bG (r = 0.974), Glucometer (r = 0.974), Chemstrip bG (r = 0.963), and Visidex (r = 0.955). For glucose values less than or equal to 180 mg/dl, Glucometer had the best correlation (r = 0.921). For glucose values of 181-400 mg/dl, Accu-Chek bG had better correlation (r = 0.907) although Glucometer had closer mean value. Visidex was just as accurate as Chemstrip bG. Although the four devices tested tended to give lower values than the lab method, they are sufficiently accurate to be of clinical use in home monitoring of blood glucose. Meticulous care in running the tests is mandatory for accuracy. Individual differences among the four methods are discussed.

The partitioning of nanoparticles to endothelium or interstitium during ultrasound-microbubble-targeted delivery depends on peak-negative pressure.

Patients diagnosed with advanced peripheral arterial disease often face poor prognoses and have limited treatment options. For some patient populations, the therapeutic growth of collateral arteries (i.e. arteriogenesis) that bypass regions affected by vascular disease may become a viable treatment option. Our group and others are developing therapeutic approaches centered on the ability of ultrasound-activated microbubbles to permeabilize skeletal muscle capillaries and facilitate the targeted delivery of pro-arteriogenic growth factor-bearing nanoparticles. The development of such approaches would benefit significantly from a better understanding of how nanoparticle diameter and ultrasound peak-negative pressure affect both total nanoparticle delivery and the partitioning of nanoparticles to endothelial or interstitial compartments. Toward this goal, using Balb/C mice that had undergone unilateral femoral artery ligation, we intra-arterially co-injected nanoparticles (50 and 100 nm) with microbubbles, applied 1 MHz ultrasound to the gracilis adductor muscle at peak-negative pressures of 0.7, 0.55, 0.4, and 0.2 MPa, and analyzed nanoparticle delivery and distribution. As expected, total nanoparticle (50 and 100 nm) delivery increased with increasing peak-negative pressure, with 50 nm nanoparticles exhibiting greater tissue coverage than 100 nm nanoparticles. Of particular interest, increasing peak-negative pressure resulted in increased delivery to the interstitium for both nanoparticle sizes, but had little influence on nanoparticle delivery to the endothelium. Thus, we conclude that alterations to peak-negative pressure may be used to adjust the fraction of nanoparticles delivered to the interstitial compartment. This information will be useful when designing ultrasound protocols for delivering pro-arteriogenic nanoparticles to skeletal muscle.

Involvement of amino acids 361 to 364 of human topoisomerase I in camptothecin resistance and enzyme catalysis.

Camptothecins are antineoplastic drugs that specifically target the enzyme DNA topoisomerase I. Prior work has identified a human topoisomerase I mutation, F361S, that confers resistance to camptothecin. We now demonstrate that substitutions in the 361-364 region can alter DNA cleavage/ligation by the enzyme. The defective catalysis exhibited by certain mutants likely relates to an impaired interaction with DNA, since these enzymes are more sensitive to the inhibitory effects of DNA binding ligands. Moreover, studies with peptides and fusion proteins suggest that the 361-364 region may bind DNA directly. The finding that the 361-364 region is involved in both enzyme catalysis and camptothecin resistance suggests that this region is part of the active site of human topoisomerase I and that camptothecin may interact with the enzyme at this site.

Gonadal function in patients with Down syndrome.

Gonadal function was evaluated in 100 home-reared persons with Down syndrome (DS) including 53 boys and men and 47 girls and women. In order to definitively evaluate gonadal function in our subjects, all patients with abnormal thyroid function were excluded from endocrine analysis. Among the men, the frequency of hypospadias and cryptorchidism was similar to that of the general population. In both men and women, the ages for the onset and completion of puberty were also normal. However, among adult men with DS, the mean stretched penile length and the mean testicular volume were significantly below the mean value of normal men. In the 23 men with DS, the mean serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were significantly elevated above the mean for normal men. By contrast, the mean plasma level of testosterone (T) was normal, suggesting a diagnosis of partial gonadal deficiency. Among the 14 women in the study, 6 had primary gonadal dysfunction. In prepubertal children, serum FSH levels in 3 boys and 5 girls were more than 2 SD above the mean for normal children, while serum levels of LH in 3 boys and 2 girls were abnormally elevated. When gonadal function was evaluated in male infants, serum levels of FSH were above the normal in 6 of 11 subjects. Serum LH was abnormally elevated in 3 of 8 female infants. Our data argue that primary gonadal deficiency is common in DS, that it is progressive from birth to adolescence, and that it is clearly manifest in adult patients.

Evidence for the reversibility of cellular DNA lesion induced by mammalian topoisomerase II poisons.

Like many intercalative antitumor drugs, the nonintercalative antitumor drug epipodophyllotoxin VM-26 (teniposide) induces topoisomerase II-linked DNA breaks as revealed by cell lysis with a strong protein denaturant such as sodium dodecyl sulfate or alkali. We show that the majority of topoisomerase II-linked DNA breaks reflect the formation of reversible topoisomerase II-DNA cleavable complexes in drug-treated cells by demonstrating the reversibility of this unusual type of DNA damage at elevated temperatures (e.g. 65 degrees C).

Comparison of various methods of blood sugar screening in newborn infants.

We evaluated two reagent strips, Dextrostix and Chemstrip-BG, and the reflectance meter technique, Stat-tek Photometer, in the screening of blood sugar values in newborn infants at high risk and compared them with laboratory glucose determination using an automatic analyzer (glucose oxidase method). The visual inspection provided by the Chemstrip-BG was the best screening method and correlated best with laboratory blood sugar concentrations, with a correlation coefficient of 0.885. This value was statistically better than for Dextrostix (r = 0.797) and Stat-tek (r = 0.777). When we compared the blood sugar values less than 50 mg/dl, the correlation coefficient for Stat-tek was 0.670, which was not statistically different than for Dextrostix (r = 0.502); Chemstrip-BG correlated best with true glucose values (r = 0.843). We believe that Chemstrip-BG is more accurate and technically easier to perform than either Dextrostix or Stat-tek.

Topoisomerase II-mediated DNA cleavage by amonafide and its structural analogs.

Treatment of SV40-infected monkey cells with amonafide (benzisoquinolinedione), an intercalative antitumor drug, resulted in rapid accumulation of linearized intracellular SV40 DNA molecules that were protein linked. Studies using purified mammalian DNA topoisomerase II have shown that amonafide and its structural analogs interfere with the breakage-rejoining reaction of the enzyme by stabilizing a reversible enzyme-DNA "cleavable complex." Denaturation of the cleavable complex with sodium dodecyl sulfate resulted in DNA cleavage and the covalent association of topoisomerase II polypeptides with the cleaved DNA. Unwinding measurements indicate that amonafide is a DNA intercalator. These results suggest that amonafide and its structural analogs (e.g., mitonafide) represent a new class of intercalative topoisomerase II-active antitumor drugs. Different from other topoisomerase II-active antitumor drugs, amonafide and mitonafide induce specific DNA cleavage at a single major site on pBR322 DNA. The strong site specificity of amonafide may allow detailed characterization of the intercalator-stabilized, topoisomerase II-DNA cleavable complex.

The role of c-Myb or a related factor in regulating the T cell receptor gamma gene enhancer.

An enhancer has been localized 3 kb downstream of the C gamma 1 gene segment of the murine TCR-gamma locus. One element, the gamma 3 site, has been shown to be critical for its functional activity. Here we have determined that Myb-related transcription factors bind to the gamma 3 site and appear to be critical for the full activity of the TCR-gamma enhancer. c-myb products can transactivate the gamma 3 site in cell lines that do not ordinarily support enhancer activity of the gamma 3 site. Mutations in the myb site or an adjacent site for core-binding factor(s) prevent transactivation. c-myb expression in various cell lines is consistent with their capacity to activate the gamma 3 enhancer element using transient transfection assays. Therefore, c-Myb or a related factor appears to play an important role in regulating the murine TCR-gamma enhancer.

Identification of a T-cell-specific transcriptional enhancer located 3' of C gamma 1 in the murine T-cell receptor gamma locus.

A transcriptional enhancer element has been localized 3 kilobases 3' of the murine T-cell receptor C gamma 1 locus using a chloramphenicol acetyltransferase reporter gene construct. As a monomer the enhancer functions only in PEER gamma delta cells and Jurkat alpha beta cells of the T-cell lines tested. However, a tetramer of the enhancer functions in virtually all T-cell lines tested, including alpha beta T-cell lines, but not in other cell types. These results suggest that elements other than the enhancer are responsible for the failure of rearranged C gamma 1 genes to be expressed in alpha beta T cells. The enhancer has been localized to a 200-base-pair Rsa I restriction fragment, which contains sequence motifs similar to those found in the other T-cell receptor enhancers but not in the immunoglobulin enhancers.