Warren Ross - Academia.edu (original) (raw)
Papers by Warren Ross
Cancer Research, 1983
The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-D-glucopy... more The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-D-glucopyranoside) , an important chemotherapeutic agent, has yet to be de termined. VP-16 has been shown to cause single-strand breaks (SSBs) in DMA, but their relationship to cytotoxicity has not been determined. We have investigated the action of VP-16 using mouse leukemia L1210 cells in culture. By using the alkaline elution technique, we reaffirmed the occurrence of SSBs in DMA over the drug concentration range 1 to 60 ¡IM. We were able to demonstrate additional types of DMA damage in the form of DMA double-strand breaks and DNA-protein cross-links within the same dose range. The number of doublestrand breaks formed per SSB was consistent over this dose range and greater than that found after exposure of L1210 cells to y-irradiation. DNA SSBs and double-strand breaks were also shown to occur in isolated nuclei, indicating that cytoplasmic components are not required for this drug action. Colony formation by L1210 cells in soft agar decreased over a drug concentration range similar to that which produced DNA damage. The correlation between the effective dose range in the colony-forming assay and the DNA scission experiments supports the hypothesis that DNA breakage is responsible for drug cytotoxicity. The demonstration of strand scission in iso lated nuclei may provide an experimental model for elucidating the exact mechanism of action of VP-16.
Cancer Research, Apr 1, 1985
The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylep... more The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylepipodophyllotoxin-9-(4,6-Oethylidene-0-D-glucopyranoside) (etoposide) (VP-16) as well as on subsequent DMA damage and cytotoxicity were studied in L1210 cells in vitro. At extracellular VP-16 concentrations of 1 to 5 UM, verapamil (10 MM) addition resulted in an increase of DNA single-strand break frequency comparable to that found when VP-16 was present alone at a 3-fold higher concentration. In addition, the elevation of cellular VP-16 levels in the presence of verapamil was linearly correlated with the enhancement of DNA damage and increased cell kill. Verapamil-mediated in crease in net VP-16 transport was rapid (1 to 2 min), and allowed for the same elevation of steady-state VP-16 concentration, whether verapamil was added simultaneously with VP-16 or was added after a steady-state level of VP-16 was achieved. Vera pamil-mediated elevation of VP-16 levels was not seen at re duced temperature (0 °C).Studies of bidirectional VP-16 trans port revealed that verapamil (40 UM)did not alter influx of VP-16 (15 UM), but lowered the unidirectional rate constant for efflux by 93%, resulting in the observed increase of steady-state level of the epipodophyllotoxin. Removal of verapamil resulted in a rapid return of VP-16 to levels comparable to that seen with VP-16 alone. When VP-16 was allowed to flow out of the cell in the presence of verapamil, less than 5% of cellular epipodophyllo toxin was retained, suggesting that the calcium antagonist is not acting by enhancing intracellular binding of VP-16. These results indicate that verapamil potentiates VP-16 activity by elevation of intracellular exchangeable epipodophyllotoxin; an activity which seems to be due to inhibition of the efflux mechanism for VP-16. The low intracellular retention of this epipodophyllotoxin and the good correlation between intracellular VP-16 and subsequent DNA damage and cytotoxicity suggest that the epipodophyllo toxin class of anticancer agents may be more useful for probing calcium antagonist effects on drug transport in sensitive cells and in cells exhibiting pleiotropic drug resistance than the vinca alkaloids and anthracyclines which have large tight binding intra cellular components.
Cancer Research, Jun 15, 1990
In an effort to shed light upon the processes of ant itunior drug-induced cell death, we have car... more In an effort to shed light upon the processes of ant itunior drug-induced cell death, we have carried out a systematic study of the effects of the anti-topoisomerase II agent, etoposide, on Chinese hamster ovary cells. Treatment of Chinese hamster ovary cells for l h with a 2-log cell-killing concentration of etoposide induces a high incidence of DNA single-strand breaks which are rapidly repaired upon drug removal. p34"u: kinase activity is inhibited within l h of addition of etoposide. Following removal of drug, cells accumulate transiently in G2. Upon recovery of p34'*; kinase activity (between 12 and 24 h posttreatment), approximately 50% of cells progress through mitosis which results in micronucleation. Ex amination of mitotic figures at various posttreatment incubation times indicates that micronucleation of daughter cells could be attributed to abnormal segregation of chromosomes during mitosis. Unexpectedly, p34<*c2 Binase activity remains elevated relative to untreated controls until 36 h post-etoposide treatment, a point where no further cell division takes place. This activity decreases by 48 h posttreatment, concomitant with a decrease in cell viability as estimated by the ability to exclude trypan blue. These results indicate that etoposide may induce cytotoxicity via gross chromosomal fragmentation, and that p34cdc2kinase may be involved in this process.
Cancer Research, Aug 1, 1984
Verapamil and a number of other Ca2+ antagonists were found to potentiate DMA damage induced by 4... more Verapamil and a number of other Ca2+ antagonists were found to potentiate DMA damage induced by 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-|3-D-glucopyranoside in L1210 cells in vitro: The potentiating effect of verapamil on DNA single-strand breaks in vitro was concentration dependent, rele vant to clinically achieved levels of Ca2+antagonists, and showed good correlation with enhanced cytotoxicity when VP-16 and Ca2+ antagonists were combined in soft agar colony-forming assays. Onset of verapamil activity was observed within 20 min of addition to cells whether VP-16 had been preincubated with cells or was added simultaneously with the Ca2+ blocker. The presence of the extracellular Ca2+ antagonist was required for potentiation as evidenced by the rapid reversal of increased DNA single-strand breaks when cells were washed free of verapamil. Neither ethyleneglycol bis(/3-aminoethyl ether)-A/,A/'-tetraacetic acid nor the Ca2+ ionophore A23187 altered verapamil potentia tion of VP-16-induced DNA damage, suggesting that this Ca2+ antagonist acts by a mechanism other than by inhibition of Ca2+ influx. In isolated L1210 nuclei, verapamil did not enhance VP-16-or 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-|8-D-glucopyranoside (VM-26)-induced single-strand breaks sug gesting a requirement for the intact cell. Even though VM-26 was 5-to 10-fold more potent than VP-16, verapamil potentiated the DNA damage caused by these two epipodophyllotoxins in L1210 cells to the same extent when these agents were used at equipotent doses. Potency differences between VM-26 and VP-16 were evident in isolated nuclei suggesting that nuclear binding or activation is a more important parameter than were previously reported membrane transport differences. The signif icance of Ca2+ antagonist potentiation of VP-16-induced DNA damage is discussed in terms of overcoming resistance to epi podophyllotoxins and characterizing more precisely the intracellular disposition, binding, and activation of VP-16.
Biochimica et biophysica acta, Jan 26, 1981
Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamyci... more Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamycin, ellipticine and actinomycin D results in DNA single-stranded breaks and DNA-protein crosslinks. To characterize further the interaction between these drugs and intracellular DNA we have employed a modification of the alkaline elution technique which allows the detection of DNA double-stranded breaks. Ellipticine (1.25-5.0 microgram/ml) adriamycin (0.5-3.0 microgram/ml) and actinomycin D (1.5-3.0 microgram/ml) all caused double-stranded breaks in DNA from L1210 cells following a 1 h treatment. The number of double-stranded breaks found per single strand break was highest for ellipticine, despite the fact that this is least cytotoxic of the three drugs. By comparing the single and double strand break frequency caused by radiation to that caused by ellipticine, it appears that most if not all of the drug-induced single strand breaks observed actually represent double-strand breaks. We su...
Cancer research, Jan 15, 1988
In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to campto... more In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisome...
Cancer treatment and research, 1991
Studies examining the mechanisms of resistance to camptothecin and its water-soluble analogs have... more Studies examining the mechanisms of resistance to camptothecin and its water-soluble analogs have been reported only recently. None of these studies have involved resistance derived in vivo in humans. Some of the mechanisms already describe could be predicted from the mechanism of action of the drug and from prior studies in yeast. It is interesting that, to date, the only mechanisms of resistance relate directly to the target of the drug, DNA topoisomerase I, and that the drugs are active in cell lines exhibiting the multidrug-resistant phenotype. Should camptothecin analogs prove as active in human clinical trials as animal tests predict, it will be interesting to see if additional mechanisms of resistance emerge from studies in treated patients. On the other hand, if clinical activity is similar to that demonstrated by camptothecin 15 years ago, the issue will be of academic interest only.
Biochimica et biophysica acta, Jan 22, 1978
The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alka... more The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alkaline elution. Adriamycin and ellipticine were found to produce large numbers of single-strand breaks. These breaks appeared to be intimately associated with protein to the extent that enzymatic deproteinization of the DNA was necessary to reveal the breaks. The frequency of breaks and cross-links increased with concentration and time of exposure to the drugs. These data suggest that DNA single-strand scission may be a feature common to intercalators. The association with a cellular protein is previously undescribed and suggests possible mechanisms for the strand scission.
Psychological Thought, 2012
This is an open access article distributed under the terms of the Creative Commons Attribution Li... more This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chromosoma, 1979
The alkaline elution technique has been modified to be used in the isolation of DNA replication i... more The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of less than 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8-12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20-30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40-50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10(6) cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.
Cancer Chemotherapy and Pharmacology, 1987
The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents ... more 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.
Cancer Chemotherapy and Pharmacology, 1989
Page 1. Cancer Chemother Pharmacol (1989)24: 167-171 ancer hemotherapy and harmacology © Springer... more Page 1. Cancer Chemother Pharmacol (1989)24: 167-171 ancer hemotherapy and harmacology © Springer-Verlag 1989 Inhibition of topoisomerases by fredericamycin A* Michael D. Latham I, Charles K. King 1, Peter Gorycki 2, Timothy L. Macdonald 2, and Warren E. Ross 1 ...
Cancer, 1983
One hundred forty-three patients with refractory cancer were treated with intensive BCNU (600-285... more One hundred forty-three patients with refractory cancer were treated with intensive BCNU (600-2850 mg/m2) and autologous marrow transplantation to determine the maximum tolerated dose and antitumor effects of this regimen. Recovery from severe pancytopenia in less than 4 weeks after transplantation occurred in 92.8% of evaluable patients, suggesting the efficacy of the autologous marrow in limiting the prolonged myelosuppression anticipated with intensive BCNU. Serious extramedullary toxicity was encountered at BCNU 1200 mg/m2, where a 9.5% incidence of fatal interstitial pneumonitis and a 3.0% incidence of fatal hepatic necrosis was observed. Higher BCNU doses, 1500 to 2850 mg/m2, were associated with a 35.3% incidence of fatal hepatotoxicity. Fatal encephalomyelopathy was encountered in two patients given BCNU 2250 and 2850 mg/m2. One patient who received the highest cumulative dose of BCNU (3450 mg/m2 in 2 courses) died of cardiac necrosis. Other serious extramedullary toxicities were not encountered, even in the 14 patients who survived from 1 to nearly 5 years after BCNU therapy. Antitumor responses occurred in 40.0% of evaluable patients; a dose effect could not be evaluated due to patient heterogeneity. The BCNU doses associated with acceptable toxicity, 600 to 1200 mg/m2, produced a 37.5% total and an 11.3% complete response (CR) rate, including five patients with prolonged CRs of 1 to nearly 5 years. Notable among the CRs was the 25.0% CR rate in previously untreated metastatic melanoma, and the production of CRs in malignant disease in the central nervous system (CNS) including melanoma, lung cancer, adenocarcinoma of unknown primary, acute leukemia and glioblastoma multiforme. It is concluded that augmented doses of BCNU can be given when autologous marrow transplantation is used to limit myelosuppression. Lung and liver toxicity prevent the use of BCNU doses greater than 1200 mg/m2; neurotoxicity, and perhaps cardiotoxicity, are manifestations of the highest doses used in this study. The antitumor activity of BCNU 600 to 1200 mg/m2 remains to be determined for most neoplasms; these results suggest improved results in melanoma and CNS malignancy compared to conventional-dose BCNU therapy.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1984
Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but... more Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05-1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and ch... more A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.
Biochemistry, 1986
Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demet... more Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.
Biochemistry, 1989
The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agen... more 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)
Biochemical Pharmacology, 1985
Abstract~This laboratory and others previously proposed that the antitumor effects of the epipodo... more Abstract~This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single-and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.
![Research paper thumbnail of Effects of serum-fractions from patients with Eaton-Lambert syndrome on rat cortical synaptosomal [3H]acetylcholine release](https://attachments.academia-assets.com/43764076/thumbnails/1.jpg)
](Biochemical Pharmacology, 1986
Eaton-Lambert syndrome is a rare disorder characterized by deficits in neuromuscular transmission... more Eaton-Lambert syndrome is a rare disorder characterized by deficits in neuromuscular transmission that result from a presynaptic inhibition of acetylcholine (ACh) release . The electrophysiological characteristics of the disease include normal miniature end-plate potential (MEPP) amplitudes, reduced number of quanta at low levels of nerve stimulation, and marked increases in the amplitude of the compound muscle action potential evoked by repetitive, supramaximal nerve stimulation [l]. Biochemically, it is known that the number of nicotinic ACh receptors [3], the ACh levels , and choline acetyltransferase [4] are unaltered in biopsied muscles from Eaton-Lambert patients. The identity and mechanism of action of the circulating inhibitor are unknown. Some neoplastic forms of the syndrome may involve the synthesis and release of neuroactive tumor-peptides, since tumor-extracts from patients with the disease can inhibit neuromuscular transmission in uitro . Alternatively, some cases of Eaton-Lambert syndrome may involve an autoimmune disorder since IgG isolated from Eaton-Lambert patients and injected into mice for several weeks can attenuate neuromuscular transmission [7] and reduce the spontaneous and electrically stimulated release of ACh from skeletal tissue
Biochemical Pharmacology, 1981
Bromoacetate, one of the hydrolysis products of bromoacetylcholine, has been shown previously to ... more Bromoacetate, one of the hydrolysis products of bromoacetylcholine, has been shown previously to inhibit the growth of neuroblastoma cells in culture. Its mechanism of action is unknown. In this work we have further characterized the cytotoxic effect of bromoacetate in C-1300 neuroblastoma cells in culture and extended it to a cell line not of neural origin, the mouse leukemia L-1210 line.
Cancer Research, 1983
The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-D-glucopy... more The precise mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-ß-D-glucopyranoside) , an important chemotherapeutic agent, has yet to be de termined. VP-16 has been shown to cause single-strand breaks (SSBs) in DMA, but their relationship to cytotoxicity has not been determined. We have investigated the action of VP-16 using mouse leukemia L1210 cells in culture. By using the alkaline elution technique, we reaffirmed the occurrence of SSBs in DMA over the drug concentration range 1 to 60 ¡IM. We were able to demonstrate additional types of DMA damage in the form of DMA double-strand breaks and DNA-protein cross-links within the same dose range. The number of doublestrand breaks formed per SSB was consistent over this dose range and greater than that found after exposure of L1210 cells to y-irradiation. DNA SSBs and double-strand breaks were also shown to occur in isolated nuclei, indicating that cytoplasmic components are not required for this drug action. Colony formation by L1210 cells in soft agar decreased over a drug concentration range similar to that which produced DNA damage. The correlation between the effective dose range in the colony-forming assay and the DNA scission experiments supports the hypothesis that DNA breakage is responsible for drug cytotoxicity. The demonstration of strand scission in iso lated nuclei may provide an experimental model for elucidating the exact mechanism of action of VP-16.
Cancer Research, Apr 1, 1985
The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylep... more The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylepipodophyllotoxin-9-(4,6-Oethylidene-0-D-glucopyranoside) (etoposide) (VP-16) as well as on subsequent DMA damage and cytotoxicity were studied in L1210 cells in vitro. At extracellular VP-16 concentrations of 1 to 5 UM, verapamil (10 MM) addition resulted in an increase of DNA single-strand break frequency comparable to that found when VP-16 was present alone at a 3-fold higher concentration. In addition, the elevation of cellular VP-16 levels in the presence of verapamil was linearly correlated with the enhancement of DNA damage and increased cell kill. Verapamil-mediated in crease in net VP-16 transport was rapid (1 to 2 min), and allowed for the same elevation of steady-state VP-16 concentration, whether verapamil was added simultaneously with VP-16 or was added after a steady-state level of VP-16 was achieved. Vera pamil-mediated elevation of VP-16 levels was not seen at re duced temperature (0 °C).Studies of bidirectional VP-16 trans port revealed that verapamil (40 UM)did not alter influx of VP-16 (15 UM), but lowered the unidirectional rate constant for efflux by 93%, resulting in the observed increase of steady-state level of the epipodophyllotoxin. Removal of verapamil resulted in a rapid return of VP-16 to levels comparable to that seen with VP-16 alone. When VP-16 was allowed to flow out of the cell in the presence of verapamil, less than 5% of cellular epipodophyllo toxin was retained, suggesting that the calcium antagonist is not acting by enhancing intracellular binding of VP-16. These results indicate that verapamil potentiates VP-16 activity by elevation of intracellular exchangeable epipodophyllotoxin; an activity which seems to be due to inhibition of the efflux mechanism for VP-16. The low intracellular retention of this epipodophyllotoxin and the good correlation between intracellular VP-16 and subsequent DNA damage and cytotoxicity suggest that the epipodophyllo toxin class of anticancer agents may be more useful for probing calcium antagonist effects on drug transport in sensitive cells and in cells exhibiting pleiotropic drug resistance than the vinca alkaloids and anthracyclines which have large tight binding intra cellular components.
Cancer Research, Jun 15, 1990
In an effort to shed light upon the processes of ant itunior drug-induced cell death, we have car... more In an effort to shed light upon the processes of ant itunior drug-induced cell death, we have carried out a systematic study of the effects of the anti-topoisomerase II agent, etoposide, on Chinese hamster ovary cells. Treatment of Chinese hamster ovary cells for l h with a 2-log cell-killing concentration of etoposide induces a high incidence of DNA single-strand breaks which are rapidly repaired upon drug removal. p34"u: kinase activity is inhibited within l h of addition of etoposide. Following removal of drug, cells accumulate transiently in G2. Upon recovery of p34'*; kinase activity (between 12 and 24 h posttreatment), approximately 50% of cells progress through mitosis which results in micronucleation. Ex amination of mitotic figures at various posttreatment incubation times indicates that micronucleation of daughter cells could be attributed to abnormal segregation of chromosomes during mitosis. Unexpectedly, p34<*c2 Binase activity remains elevated relative to untreated controls until 36 h post-etoposide treatment, a point where no further cell division takes place. This activity decreases by 48 h posttreatment, concomitant with a decrease in cell viability as estimated by the ability to exclude trypan blue. These results indicate that etoposide may induce cytotoxicity via gross chromosomal fragmentation, and that p34cdc2kinase may be involved in this process.
Cancer Research, Aug 1, 1984
Verapamil and a number of other Ca2+ antagonists were found to potentiate DMA damage induced by 4... more Verapamil and a number of other Ca2+ antagonists were found to potentiate DMA damage induced by 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-|3-D-glucopyranoside in L1210 cells in vitro: The potentiating effect of verapamil on DNA single-strand breaks in vitro was concentration dependent, rele vant to clinically achieved levels of Ca2+antagonists, and showed good correlation with enhanced cytotoxicity when VP-16 and Ca2+ antagonists were combined in soft agar colony-forming assays. Onset of verapamil activity was observed within 20 min of addition to cells whether VP-16 had been preincubated with cells or was added simultaneously with the Ca2+ blocker. The presence of the extracellular Ca2+ antagonist was required for potentiation as evidenced by the rapid reversal of increased DNA single-strand breaks when cells were washed free of verapamil. Neither ethyleneglycol bis(/3-aminoethyl ether)-A/,A/'-tetraacetic acid nor the Ca2+ ionophore A23187 altered verapamil potentia tion of VP-16-induced DNA damage, suggesting that this Ca2+ antagonist acts by a mechanism other than by inhibition of Ca2+ influx. In isolated L1210 nuclei, verapamil did not enhance VP-16-or 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-|8-D-glucopyranoside (VM-26)-induced single-strand breaks sug gesting a requirement for the intact cell. Even though VM-26 was 5-to 10-fold more potent than VP-16, verapamil potentiated the DNA damage caused by these two epipodophyllotoxins in L1210 cells to the same extent when these agents were used at equipotent doses. Potency differences between VM-26 and VP-16 were evident in isolated nuclei suggesting that nuclear binding or activation is a more important parameter than were previously reported membrane transport differences. The signif icance of Ca2+ antagonist potentiation of VP-16-induced DNA damage is discussed in terms of overcoming resistance to epi podophyllotoxins and characterizing more precisely the intracellular disposition, binding, and activation of VP-16.
Biochimica et biophysica acta, Jan 26, 1981
Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamyci... more Previous work has shown that exposing mouse L1210 cells to intercalating agents such as adriamycin, ellipticine and actinomycin D results in DNA single-stranded breaks and DNA-protein crosslinks. To characterize further the interaction between these drugs and intracellular DNA we have employed a modification of the alkaline elution technique which allows the detection of DNA double-stranded breaks. Ellipticine (1.25-5.0 microgram/ml) adriamycin (0.5-3.0 microgram/ml) and actinomycin D (1.5-3.0 microgram/ml) all caused double-stranded breaks in DNA from L1210 cells following a 1 h treatment. The number of double-stranded breaks found per single strand break was highest for ellipticine, despite the fact that this is least cytotoxic of the three drugs. By comparing the single and double strand break frequency caused by radiation to that caused by ellipticine, it appears that most if not all of the drug-induced single strand breaks observed actually represent double-strand breaks. We su...
Cancer research, Jan 15, 1988
In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to campto... more In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisome...
Cancer treatment and research, 1991
Studies examining the mechanisms of resistance to camptothecin and its water-soluble analogs have... more Studies examining the mechanisms of resistance to camptothecin and its water-soluble analogs have been reported only recently. None of these studies have involved resistance derived in vivo in humans. Some of the mechanisms already describe could be predicted from the mechanism of action of the drug and from prior studies in yeast. It is interesting that, to date, the only mechanisms of resistance relate directly to the target of the drug, DNA topoisomerase I, and that the drugs are active in cell lines exhibiting the multidrug-resistant phenotype. Should camptothecin analogs prove as active in human clinical trials as animal tests predict, it will be interesting to see if additional mechanisms of resistance emerge from studies in treated patients. On the other hand, if clinical activity is similar to that demonstrated by camptothecin 15 years ago, the issue will be of academic interest only.
Biochimica et biophysica acta, Jan 22, 1978
The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alka... more The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alkaline elution. Adriamycin and ellipticine were found to produce large numbers of single-strand breaks. These breaks appeared to be intimately associated with protein to the extent that enzymatic deproteinization of the DNA was necessary to reveal the breaks. The frequency of breaks and cross-links increased with concentration and time of exposure to the drugs. These data suggest that DNA single-strand scission may be a feature common to intercalators. The association with a cellular protein is previously undescribed and suggests possible mechanisms for the strand scission.
Psychological Thought, 2012
This is an open access article distributed under the terms of the Creative Commons Attribution Li... more This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chromosoma, 1979
The alkaline elution technique has been modified to be used in the isolation of DNA replication i... more The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of less than 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8-12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20-30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40-50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10(6) cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.
Cancer Chemotherapy and Pharmacology, 1987
The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents ... more 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.
Cancer Chemotherapy and Pharmacology, 1989
Page 1. Cancer Chemother Pharmacol (1989)24: 167-171 ancer hemotherapy and harmacology © Springer... more Page 1. Cancer Chemother Pharmacol (1989)24: 167-171 ancer hemotherapy and harmacology © Springer-Verlag 1989 Inhibition of topoisomerases by fredericamycin A* Michael D. Latham I, Charles K. King 1, Peter Gorycki 2, Timothy L. Macdonald 2, and Warren E. Ross 1 ...
Cancer, 1983
One hundred forty-three patients with refractory cancer were treated with intensive BCNU (600-285... more One hundred forty-three patients with refractory cancer were treated with intensive BCNU (600-2850 mg/m2) and autologous marrow transplantation to determine the maximum tolerated dose and antitumor effects of this regimen. Recovery from severe pancytopenia in less than 4 weeks after transplantation occurred in 92.8% of evaluable patients, suggesting the efficacy of the autologous marrow in limiting the prolonged myelosuppression anticipated with intensive BCNU. Serious extramedullary toxicity was encountered at BCNU 1200 mg/m2, where a 9.5% incidence of fatal interstitial pneumonitis and a 3.0% incidence of fatal hepatic necrosis was observed. Higher BCNU doses, 1500 to 2850 mg/m2, were associated with a 35.3% incidence of fatal hepatotoxicity. Fatal encephalomyelopathy was encountered in two patients given BCNU 2250 and 2850 mg/m2. One patient who received the highest cumulative dose of BCNU (3450 mg/m2 in 2 courses) died of cardiac necrosis. Other serious extramedullary toxicities were not encountered, even in the 14 patients who survived from 1 to nearly 5 years after BCNU therapy. Antitumor responses occurred in 40.0% of evaluable patients; a dose effect could not be evaluated due to patient heterogeneity. The BCNU doses associated with acceptable toxicity, 600 to 1200 mg/m2, produced a 37.5% total and an 11.3% complete response (CR) rate, including five patients with prolonged CRs of 1 to nearly 5 years. Notable among the CRs was the 25.0% CR rate in previously untreated metastatic melanoma, and the production of CRs in malignant disease in the central nervous system (CNS) including melanoma, lung cancer, adenocarcinoma of unknown primary, acute leukemia and glioblastoma multiforme. It is concluded that augmented doses of BCNU can be given when autologous marrow transplantation is used to limit myelosuppression. Lung and liver toxicity prevent the use of BCNU doses greater than 1200 mg/m2; neurotoxicity, and perhaps cardiotoxicity, are manifestations of the highest doses used in this study. The antitumor activity of BCNU 600 to 1200 mg/m2 remains to be determined for most neoplasms; these results suggest improved results in melanoma and CNS malignancy compared to conventional-dose BCNU therapy.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1984
Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but... more Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05-1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1983
A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and ch... more A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.
Biochemistry, 1986
Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demet... more Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.
Biochemistry, 1989
The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agen... more 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)
Biochemical Pharmacology, 1985
Abstract~This laboratory and others previously proposed that the antitumor effects of the epipodo... more Abstract~This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single-and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.
Biochemical Pharmacology, 1986
Eaton-Lambert syndrome is a rare disorder characterized by deficits in neuromuscular transmission... more Eaton-Lambert syndrome is a rare disorder characterized by deficits in neuromuscular transmission that result from a presynaptic inhibition of acetylcholine (ACh) release . The electrophysiological characteristics of the disease include normal miniature end-plate potential (MEPP) amplitudes, reduced number of quanta at low levels of nerve stimulation, and marked increases in the amplitude of the compound muscle action potential evoked by repetitive, supramaximal nerve stimulation [l]. Biochemically, it is known that the number of nicotinic ACh receptors [3], the ACh levels , and choline acetyltransferase [4] are unaltered in biopsied muscles from Eaton-Lambert patients. The identity and mechanism of action of the circulating inhibitor are unknown. Some neoplastic forms of the syndrome may involve the synthesis and release of neuroactive tumor-peptides, since tumor-extracts from patients with the disease can inhibit neuromuscular transmission in uitro . Alternatively, some cases of Eaton-Lambert syndrome may involve an autoimmune disorder since IgG isolated from Eaton-Lambert patients and injected into mice for several weeks can attenuate neuromuscular transmission [7] and reduce the spontaneous and electrically stimulated release of ACh from skeletal tissue
Biochemical Pharmacology, 1981
Bromoacetate, one of the hydrolysis products of bromoacetylcholine, has been shown previously to ... more Bromoacetate, one of the hydrolysis products of bromoacetylcholine, has been shown previously to inhibit the growth of neuroblastoma cells in culture. Its mechanism of action is unknown. In this work we have further characterized the cytotoxic effect of bromoacetate in C-1300 neuroblastoma cells in culture and extended it to a cell line not of neural origin, the mouse leukemia L-1210 line.