Biomedical subjects

J Maybaum

Publications and source records attributed to J Maybaum.

At least 19 recordsLinked to original sources

PubMed1992

Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity.

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.

Cell Survival↗

PubMed1992

The potential superiority of bromodeoxyuridine to iododeoxyuridine as a radiation sensitizer in the treatment of colorectal cancer.

Although the thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) and 5-iodo-2'-deoxyuridine (IdUrd) have been used successfully as radiation sensitizers in clinical trials, it is not clear which of these agents is the more promising to pursue. To begin to assess this question with regard to colorectal cancer metastatic to the liver, a study was carried out using HT29 human colon cancer cells in culture and implanted in nude mice as xenografts. Cells and animals were treated with BrdUrd +/- the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd), and the results compared to our previous studies with IdUrd +/- FdUrd (T. S. Lawrence, M. A. Davis, P. E. McKeever, J. Maybaum, P. L. Stetson, D. P. Normolle, and W. D. Ensminger. Cancer Res., 51: 3900-3905, 1991). Using cultured cells, it was found that FdUrd (at concentrations of greater than 10 nM) increased: (a) the incorporation of BrdUrd into the DNA of cultured tumor cells; (b) BrdUrd-mediated radiosensitization; (c) BrdUrd-mediated increase in radiation-induced DNA damage; and (d) BrdUrd-mediated decrease in the repair of radiation-induced damage. The incorporation of BrdUrd was greater than or equal to the incorporation of IdUrd previously determined under the same exposure conditions. Studies using nude mice bearing HT29 xenografts showed that FdUrd increased BrdUrd incorporation more into tumors than into the normal liver. Most tumor cells incorporated BrdUrd (labeling index after a 4-day infusion = 87 +/- 2%; SE); in the liver, labeling was confined chiefly to nonparenchymal cells. In both the presence and absence of FdUrd, the incorporation of BrdUrd into tumors was significantly and consistently greater than the incorporation of IdUrd measured under the same conditions of drug administration (by a factor of 1.2-3.6). Furthermore, the administration of BrdUrd +/- FdUrd tended to produce less weight loss and hematological toxicity than IdUrd +/- FdUrd. These findings suggest that BrdUrd may be superior to IdUrd as a radiation sensitizer in the treatment of colorectal cancer metastatic to the liver.

Animals↗

PubMed1992

Modulation of iododeoxyuridine-mediated radiosensitization by 5-fluorouracil in human colon cancer cells.

The incorporation of 5-iodo-2'-deoxyuridine (IdUrd), a thymidine analog radiosensitizer, can be increased by the use of modulators such as 5-fluorouracil (FUra). FUra is a particularly attractive potential modulator to use against colorectal cancer, as it is the most active single agent in the treatment of this disease. To begin to define the conditions for the optimal combination of IdUrd and FUra in the treatment of patients with colorectal cancer, a study was conducted of the effect of FUra on IdUrd-mediated radiosensitization in cultured HT29 human colon cancer cells. It was found that when cells were exposed to concentrations of IdUrd typical of those obtained through intravenous exposure (1-3 microM), FUra (1 microM) increased radiosensitization beyond that which would be predicted for the same extent of incorporation produced by incubation with IdUrd alone. This increase appeared to result from a combination of at least two effects: FUra-mediated cell cycle redistribution and increased IdUrd incorporation. When a higher concentration of IdUrd (10 microM) was used with FUra (1 microM), cell cycle distribution returned to nearly normal, and radiosensitization was equal to that predicted by the extent of incorporation of IdUrd. These data demonstrate that the combination of FUra and IdUrd can produce radiosensitization both through increased IdUrd incorporation and cell cycle redistribution. Furthermore, they suggest that, in the presence of a modulator, it may not be necessary to achieve high levels of IdUrd incorporation to produce significant tumor radiosensitization.

Cell Cycle↗

PubMed1991

Fluorodeoxyuridine-mediated modulation of iododeoxyuridine incorporation and radiosensitization in human colon cancer cells in vitro and in vivo.

A study was conducted to assess the potential of 5-fluoro-2'-deoxyuridine (FdUrd) to increase the incorporation and radiosensitizing properties of 5-iodo-2'-deoxyuridine (IdUrd) using HT29 human colon cancer cells both in vitro and in nude mice bearing these tumors as xenografts. The purpose of this study was to assess (a) whether FdUrd could increase IdUrd efficacy using clinically achievable concentrations of drugs; (b) the relationships among radiosensitization, DNA damage and repair, and analogue incorporation; and (c) whether FdUrd improved the selectivity of IdUrd incorporation into tumor cells compared to normal tissues. It was found that FdUrd, at clinically achievable concentrations (1-100 nM), significantly increased IdUrd incorporation under all conditions but particularly when the IdUrd concentration was less than or equal to 10 microM. FdUrd increased IdUrd-mediated radiosensitization in proportion to the increase in IdUrd incorporation. FdUrd potentiated the ability of IdUrd to increase radiation-induced DNA double-strand breaks and to slow their repair. When IdUrd alone (100 and 200 mg/kg/day) was infused into nude mice bearing tumors, the extent of thymidine replaced in the tumor was 1.6 +/- 0.4 (mean +/- SE) and 2.5 +/- 0.4%, respectively. The combination of FdUrd (0.1 mg/kg/day) and IdUrd (100 mg/kg/day) increased the incorporation in the tumor to 5.3 +/- 0.9% with less toxicity than resulted from the use of 200 mg/kg/day of IdUrd alone. These data show that FdUrd is an effective biomodulator, because, for the same extent of normal tissue incorporation, the combination of IdUrd and FdUrd produces significantly greater incorporation into the tumor compared to the use of IdUrd alone. Furthermore, they suggest that the regional application of FdUrd with IdUrd, either through the use of regional infusions or in combination with focused irradiation, could potentially improve the outcome of treatment of localized gastrointestinal cancer.

Animals↗

PubMed1991

Divergent patterns of incorporation of bromodeoxyuridine and iododeoxyuridine in human colorectal tumor cell lines.

Using a panel of four human colorectal tumor (HCT) cell lines, we have quantitatively characterized the incorporation of bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) into DNA, both as individual agents and in combination with fluoropyrimidines. The intrinsic ability of these cell lines to incorporate BrdUrd, as reflected by the concentration required to achieve half-maximal incorporation, varied almost 4-fold across this panel, from 1.6 microM for HuTu80 cells to 6.1 microM for HT29 cells. Three of the four cell lines (HT29, SW480, SW620) responded to fluoropyrimidines as expected, displaying 100-150% increases in BrdUrd incorporation when combined with growth inhibitory concentrations of fluorouracil (FUra). In contrast, neither FUra nor fluorodeoxyuridine (FdUrd) was able to increase BrdUrd incorporation in HuTu80 cells by more than 25%, even in the presence of 100 microM leucovorin. IdUrd incorporation was modulated to a substantially higher degree in both HT29 and HuTu80 cell lines. Finally we demonstrate the feasibility of a technique for evaluating the net effect of fluoropyrimidine treatments on de novo thymidine nucleotide production in a single specimen, using a combination of normotopic and stable-isotope labeled BrdUrd. We propose that this approach may be useful in evaluating the response of an individual tumor to fluoropyrimidines in vivo.

Bromodeoxyuridine↗

PubMed1991

Induction of megabase DNA fragments by 5-fluorodeoxyuridine in human colorectal tumor (HT29) cells.

Current evidence suggests that DNA fragmentation plays an integral role in mediating cytotoxicity that results from thymidine nucleotide depletion ("thymineless death"). Recently, Ayusawa et al. [Mutat. Res. 200:221-230 (1988)] reported that dTMP starvation induces cellular processes that result in the release of 50-200-kilobase (kb) DNA fragments in FM3A cells, as detected by pulsed field gel electrophoresis (PFGE). The present study was undertaken to determine whether a similar DNA fragmentation process occurs in a human cell line in response to fluoropyrimidine treatment and, if so, to quantitate this process. When human colorectal tumor (HT29) cells were treated with 100 nm 5-fluorodeoxyuridine (FdUrd), this regimen induced the formation of high molecular weight DNA fragments, which were analyzed using three different PFGE protocols. Field inversion PFGE revealed that, in contrast to the discrete size range reported for FM3A cells, FdUrd-induced fragments in HT29 cells were broadly distributed from about 50 kb to sizes beyond the resolution of the field inversion mode (i.e., greater than 600 kb). Analysis of these same samples by contour-clamped homogeneous electric field PFGE showed that the bulk of these fragments migrated in the 1-5-megabase region. In contrast, fragments from DNA that was broken randomly by gamma-radiation appeared to be primarily in a zone corresponding to approximately 5-10 megabases. Equitoxic FdUrd and radiation treatments (100 nM FdUrd for 48 hr versus 10-Gy gamma-radiation) each increased the fraction of DNA entering the gel from about 0.07-0.09 (untreated cells) to about 0.22-0.25. To a first approximation, the time course and quantity of DNA fragmentation induced by 100 nM FdUrd appeared to correlate with the loss of clonogenicity within the 48-hr period analyzed. These findings suggest that the processes responsible for DNA fragmentation in response to a thymineless state may be different in FM3A and HT29 cells, that in both cases the breaks caused do not appear to be located randomly with respect to the entire genome, and that these processes may be related to the chain of events by which temporary dTMP starvation is made into a lethal insult.

Cell Survival↗

PubMed1991

Selective inhibition of sequence-specific protein-DNA interactions by incorporation of 6-thioguanine: cleavage by restriction endonucleases.

Incorporation of the antileukemic agent 6-thioguanine (TG) into cellular DNA has been demonstrated to be a major determinant of its cytotoxicity. We have previously shown that complete replacement of G by TG within one DNA strand of the SV40 origin of replication can completely inhibit sequence-specific binding of the viral replication protein T antigen. The aim of the present study was to determine the effect of more selective TG substitutions on DNA-protein interactions, by utilizing the simpler base recognition sequence motifs of restriction endonucleases. In the first part of our study, we replaced G with TG in one or two of four possible sites within the duplex hexameric recognition sequence of BamHI (5'-G decreases GATCC-3'), by enzymatic extension of primed oligonucleotides. This extension was stalled, but not completely inhibited, at locations where insertion of consecutive TG moieties was required. Both strands of molecules containing a single substitution were cleaved by BamHI at reduced rates, with the substituted strand inhibited to a greater degree. In molecules containing two substitutions, neither strand was cut by BamHI. In contrast, we found that scission of these same mono- and disubstituted substrates by the less stringent isoschizomer MboI (5'-N decreases GATCN-3') was inhibited only slightly. In the second part of our study, we investigated the effect of analog substitution on scission by the type II-S enzymes AlwI and FokI, in order to separately determine the effects of restriction site modification versus scission site modification. We found that the reactivity of these enzymes was completely abolished by TG substitution within the recognition site, whereas substitution at the scission site had no effect. Our results demonstrate that infrequent TG substitutions within symmetric DNA sequences can inhibit sequence-specific interactions in an asymmetric fashion. In addition, although previous reports have shown that TG forms a relatively weak base pair with cytosine, it appears that the inhibition of restriction endonuclease-mediated cleavage resulting from TG incorporation is a function of the sequence requirements of the protein and not a general consequence of disrupted base-pairing at the recognition locus. These data support the idea that the cytotoxic consequences of TG incorporation may be due to inhibition of sequence-specific protein-DNA interactions.

Base Sequence↗

PubMed1990

Modulation of the cytotoxic mechanism of 6-thioguanine by 4-amino-5-imidazolecarboxamide.

Previous evidence has indicated that either purine starvation or incorporation into DNA may be the dominant biochemical effect of the antileukemic agent 6-thioguanine (TG), depending on exposure conditions. Furthermore, it has been suggested that the paradoxical decrease in TG-induced cytotoxicity at high drug concentrations may be due to an antagonistic interaction between these two mechanisms, in which purine starvation inhibits DNA synthesis and, therefore, incorporation of TG into DNA. In this report we test the hypothesis that by concurrent treatment of L1210 cells with TG and the purine precursor 4-amino-5-imidazolecarboxamide (AIC) it is possible to alleviate DNA synthesis inhibition caused by high concentrations of TG, thus enhancing TG incorporation into DNA and TG-induced cell kill. Both the cytotoxic and cytokinetic results presented support this hypothesis. However, gross incorporation of TG into DNA was not increased by AIC under conditions in which a significant enhancement of cytotoxicity (i.e., 1 log) was observed. These findings suggest that the potentiating effect of AIC may be most prominent on the subpopulation of cells that are resistant to treatment with TG alone, and they demonstrate that the cytotoxic effects of TG treatments are more accurately reflected by observing specific cytokinetic changes (delayed late S/G2 arrest) than by measuring the average extent of TG incorporation into DNA within a given population. Finally, we propose that it may be possible to select conditions for administration of TG that favor one or the other cytotoxic mechanism, depending on whether the clinical objective is induction of remission (where rapid cell lysis due to purine starvation would be desired) or eradication of subclinical disease during remission (where proliferation-dependent cytotoxicity due to DNA incorporation should be more effective.

Aminoimidazole Carboxamide↗

PubMed1990

The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure.

The influence of the duration of exposure to the halogenated pyrimidines iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) on incorporation into DNA and the resulting radiosensitization was studied in cultured human colon cancer cells. Cells were incubated with either 10 microM BrdUrd or IdUrd for periods up to 7 days. They were also assessed for up to 4 days after removal of drug from the medium. Replacement of thymidine by fraudulent bases was measured using a sensitive gas chromatographic, mass spectrometric (GC/MS) assay. Incorporation of BrdUrd and IdUrd plateaued at 35% and 30%, respectively, after 4 days of exposure. Prolonging the time of exposure to 7 days increased cytotoxicity without affecting either incorporation or radiosensitization. Incorporation remained constant for 1-2 days after removal of drug from the medium. Radiosensitization was linearly related to incorporation throughout the range of conditions assessed. These data suggest that it may be possible to develop a predictive assay for radiosensitization based on measurements of halogenated pyrimidine incorporation in a tumor biopsy specimen. They also suggest that a clinical approach based on repeated short exposures to halogenated pyrimidines may present certain advantages over the current practice of prolonged continuous exposure. A Phase I/II trial using IdUrd and external beam irradiation for the treatment of patients with poor prognosis soft tissue sarcomas has been initiated based on this concept.

Bromodeoxyuridine↗

PubMed1990

The effect of single versus double-strand substitution on halogenated pyrimidine-induced radiosensitization and DNA strand breakage in human tumor cells.

To better understand the mechanism underlying halogenated pyrimidine-mediated cytotoxicity and radiosensitization in human tumor cells, a study was undertaken to determine the influence of unifilar (one DNA strand) versus bifilar (both DNA strands) substitution of thymidine by the halogenated bases 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) in HT29 human colon cancer cells. Unifilar labeling was obtained by incubating cells with IdUrd or BrdUrd for one doubling time. Cells were incubated for at least three doublings to approximate bifilar substitution. Only IdUrd caused significant cytotoxicity, which correlated with incorporation into DNA. Both BrdUrd and IdUrd were potent radiosensitizers. Radiosensitization was linearly correlated with incorporation of both bases regardless of the number of strands in which thymidine was substituted. In contrast, the relationship between radiosensitization and DNA double-strand breakage was critically dependent in the case of IdUrd, but not for BrdUrd, on whether substitution was unifilar or bifilar. These findings suggest that incorporation is the best predictor of radiation sensitivity, and that the induction of DNA double-strand breaks alone does not account for radiosensitization mediated by halogenated pyrimidines in these human tumor cells.

Bromodeoxyuridine↗

PubMed1989

Dependence of etoposide-induced cytotoxicity and topoisomerase II-mediated DNA strand breakage on the intracellular ionic environment.

We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.

Animals↗

PubMed1989

Prediction of thioguanine-induced cytotoxicity by dual-parameter flow cytometric analysis.

A method is presented for the quantitative analysis of delayed cytokinetic effects resulting from the treatment of L1210 cells with 6-thioguanine (TG). By using dual-parameter (DNA/protein) flow cytometry, we could observe the accumulation of late S/G2/M cells with abnormally high green fluorescence (i.e., protein content), indicative of unbalanced growth. The use of mitotic cells from a pseudotetraploid line (HT29) as external markers for both red and green fluorescence facilitated highly reproducible measurement of the mean green fluorescence (GFLmean) of the arrested late S/G2/M population. We found that the dose dependence of the observed GFLmean values followed the same unusual biphasic pattern as did cytotoxicity in this cell line, indicating that this parameter might be a suitable means of predicting TG-induced toxicity in vivo. We propose that the low background expected for this kind of measurement would make it particularly appropriate for the analysis of clinical specimens (e.g., mononuclear bone marrow cells) from leukemic patients receiving thiopurines, to monitor (and, hopefully, predict) their response to treatment.

Algorithms↗

PubMed1989

Response of human HT-29 colorectal tumor cells to extended exposure to bromodeoxyuridine.

Effects of the extended exposure of a human colorectal tumor-cell line (HT-29) to bromodeoxyuridine (BrdUrd) were studied in anticipation of the clinical use of that agent to treat colorectal cancer, particularly as a regionally delivered radiosensitizer. We found that 72-h exposure to a concentration of BrdUrd that is estimated to be locally maintained in the liver (100 microM) was significantly cytotoxic with a 3-log reduction in survival. As measured by GC/MS-SIM method, incorporation of BrdUrd into DNA followed an unexpected time course in that continuous exposure to 10 microM BrdUrd resulted in maximal incorporation at 3 days, after which the extent of incorporated analog fell significantly (despite daily changes of the medium). This finding was apparently due to a greater rate of loss of BrdUrd from the medium at later time points. Flow cytometric analysis using an anti-BrdUrd antibody (IU-4) revealed that antibody binding also peaked and fell off with time. However, at exposure times of greater than 24 h, the timing and extent of this decline were significantly different than had been indicated by the GC/MS method. These results indicate that the quantitative relationship between antibody staining and BrdUrd incorporation changes as drug-exposure time increases and that quantitative studies of anti-BrdUrd antibody binding must be interpreted with caution, especially when extended drug-treatment protocols have been used.

Bromodeoxyuridine↗

PubMed1988

Tissue-specific pharmacodynamics of 5-bromo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.

The thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd) is felt to exert its cytotoxic effects primarily through incorporation into DNA. We have evaluated the incorporation of BrdUrd into the DNA of relevant normal tissues (bone marrow, gut mucosa, and liver) and tumor in rabbits with the VX2 tumor growing intrahepatically. Using constant i.v. infusions, steady state plasma drug concentrations ranging from 0.4 to 65.4 microM were maintained for 24 h and tissues were harvested and processed so that a sensitive gas chromatography/mass spectrometry (GC/MS) method could be used to analyze the thymine and 5-bromouracil content of hydrolyzed DNA. In all tissues, DNA incorporation showed saturating effects as plasma BrdUrd concentration was increased and, BrdUrd incorporation as a function of plasma concentration could be fitted to a Langmuir-like equation generating tissue-specific pharmacodynamic parameters: Imax for percentage thymine replacement at infinite plasma BrdUrd concentrations, and C50 for the arterial BrdUrd concentration generating incorporation that is Imax/2. At all plasma concentrations of BrdUrd the incorporation into DNA of bone marrow was greater than that observed in VX2 tumor. However, BrdUrd labeling index (with a BrdUrd monoclonal antibody) was greater in tumor than bone marrow. Thus, pharmacodynamic differences in incorporation do not result solely from cytokinetic differences between tissues. This model may prove useful in evaluating the pharmacodynamics of incorporation in studies using hepatic arterial infusion and biochemical modulation to improve selectivity.

Animals↗

PubMed1988

Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity.

Inhibitors of DNA methylation, such as 5-azacytidine, induce gene expression. We have previously reported that cloned T cells treated with 5-azacytidine lose the requirement for Ag and can be activated by autologous HLA-D molecules alone, thus becoming auto-reactive. This phenomenon could potentially mediate an autoimmune disease in vivo. Inasmuch as several drugs are known to cause autoimmune disease, we asked whether they exert the same effects on T cells as 5-azacytidine. We report that hydralazine and procainamide, two drugs associated with a lupus-like autoimmune disease, also inhibit DNA methylation and induce self-reactivity in cloned T cell lines. These results suggest that drug-induced autoimmune disease may be due to activation of as yet unidentified genes through mechanisms involving DNA methylation.

Autoimmune Diseases↗

PubMed1988

N-acetylprocainamide is a less potent inducer of T cell autoreactivity than procainamide.

We have reported that an inhibitor of DNA methylation, 5-azacytidine, makes cloned, antigen-specific CD4+ T cells autoreactive, and that procainamide and hydralazine mimic this effect. Those results suggested that procainamide and hydralazine may induce autoimmunity by inhibiting DNA methylation and causing T cell autoreactivity. We report now that N-acetylprocainamide, a procainamide derivative that does not induce lupus, is also a DNA methylation inhibitor, but it is 100 times less potent than procainamide in inducing T cell autoreactivity.

Acecainide↗

PubMed1988

Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture.

By inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G1 phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX), we examined the hypothesis that this modulation can allow normal rates of RNA synthesis and cell cycle progression from G1 to S phase. For L1210 cells exposed to MTX for 12 h or 24 h, the addition of HX enhanced the cytotoxicity of MTX; however, no enhancement was observed with a 6-h exposure. Inhibition of RNA synthesis by MTX was prevented by concurrent administration of HX. The effect of HX on cell cycle progression was first examined using flow cytometry, which indicated that MTX treatment alone or with concurrent HX caused a buildup of cells with a G1 content of DNA. Because this technique may fail to distinguish between cells in late G1 phase, the G1/S border, or early S, the method of premature chromosome condensation was used to determine cell cycle position based on chromatin morphology. A shift to a higher degree of chromatin decondensation was observed when HX was coadministered with MTX during a 12-h exposure, suggesting progression from G1 towards S. This correlated with the enhancement of MTX cytotoxicity by HX after 12 h exposure. The results of these studies suggest that HX potentiates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that might ordinarily be arrested in G1 to progress into the cytotoxic S phase.

Animals↗

PubMed1988

Dynamics of bromodeoxyuridine incorporation into DNA of squamous carcinoma cells during mid and late logarithmic growth.

Head and neck squamous carcinoma cell lines, UM-SCC 1, 5, 9, 11B, and 14B, were exposed in vitro to bromodeoxyuridine (BUdR) during logarithmic growth to determine the effects of drug concentration (0.01 to 10 microM) and duration of exposure (3, 7, and 10 days) on cell growth and on incorporation of BUdR into DNA. Concentrations of less than 1.0 microM were not growth inhibitory except with UM-SCC-11B. After 10 days of exposure to 5 microM BUdR, survival fractions for all lines ranged from 2 to 65% of controls. Replacement of thymidine by BUdR in DNA was assessed by gas chromatography/mass spectroscopy. Percentage replacement (% R) was described by the equation % R = 100 (C/t)s/[(C/t)50s + (C/t)s], where C is the concentration of BUdR (microM), t is the time in days, s is a constant, and (C/t)50 is a constant corresponding to % R = 50%. BUdR incorporation reached a time- and concentration-dependent maximum that, after 3 to 7 days of culture in 10 microM BUdR, ranged from 30 to 60% R. Subsequently, % R declined with time even though the cells were fed daily with fresh BUdR-containing medium.

Bromodeoxyuridine↗