Low and high concentrations of the topo II inhibitor daunorubicin in NIH3T3 cells: reversible G2/M versus irreversible G1 and S arrest

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DMA. In both drug-sensitive 3T3 cells and drug-resistant 3T3 cells (NIH-MDR-6185, created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells) initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells, and accumulated in G(2)/M. The 2 to 3 h lag period represents the time taken for fully establishing the G(2)/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G] and S, and did not reach G(2)/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G(2)/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).     

Effects of cytosine arabinoside and daunorubicin on survival and cell cycle progression of chinese hamster ovary cells

Abstract. Kinetic and cytotoxic effects of cytosine arabinoside (Ara-C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony-forming assay, respectively. With Ara-C alone, increasing drug concentrations between 10^-7 M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10^-4 M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 times 10^-7 M. It decreased the rate of progression through S phase and caused cells to accumulate in G₂, except at the highest concentration (2 times 10^-5 M), at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara-C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non-lethal dose of Ara-C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug-free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by druginduced redistribution of cells within the cell cycle, but may have been related to the effects of the non-lethal drug on DNA synthesis rate.     

Serum-stimulated 3T3 cells undertake a histidinol-sensitive process which G1 cells do not

Serum-stimulated Swiss 3T3 cells previously arrested by either serum starvation or high cell density undergo some process(es) sensitive to low concentrations of histidinol, whereas G1 cells transiting from M to S do not. The inhibition of transit to S induced by histidinol is reversible.     

Continuous protein synthesis is required to maintain the probability of entry into S phase

“Normal fibroblast” lines such as 3T3 cells arrest in the G0/G1 compartment of the cell cycle when starved of serum. Following readdition of serum and after a lag of 14 hr, the cells enter S phase with first-order kinetics. Cell cycle progress after stimulation is thus consistent with the existence of a single, rate-limiting random event (or transition) in G1 as proposed by Smith and Martin (1973). The addition of low concentrations of cycloheximide (33–100 ng/ml) at any time after the end of the lag phase brings about a rapid reduction (within 1–2 hr) of the rate constant for entry into S phase by an amount that is proportional to the inhibition of leucine incorporation. This suggests that the transition probability depends upon the continuous synthesis of a protein with a short half-life, or on some other unstable substance whose concentration is geared to the rate of translation. More importantly at present, the results indicate that the rate-limiting transition occurs within 2 hr of the start of DNA synthesis.When the same low concentrations of cycloheximide are added at the time of serum stimulation, they also lead to a marked elongation of the lag phase which again is related to the inhibitor concentration. This result is surprising since the lag is independent of serum concentration which itself influences the rate of protein synthesis.     

The concentration-dependent diversity of effects of DNA topoisomerase I and II inhibitors on the cell cycle of HL-60 cells.

Exposure of promyelocytic leukemic HL-60 cells to 3-60 nM of the DNA topoisomerase I inhibitor camptothecin (CAM) or to 30-450 nM and 0.12-1.5 microM of DNA topoisomerase II inhibitors teniposide (TN) and 4-(9-acridynylamino)-3-methanesulfon-m-anisidide (m-AMSA), respectively, resulted in two distinct kinetic effects: (1) the cells entered S phase but the rate of DNA replication was reduced in proportion to the inhibitor concentration; (2) the transition from G2 to M was impaired, approximately 1 h after addition of the inhibitor. As a consequence, the cells accumulated in the S (preferentially in early S) and in G2 phases of the cell cycle. Whereas CAM was more efficient in suppressing cell progression through S phase, TN and m-AMSA were more potent G2 blockers. At these low inhibitor concentrations no signs of immediate cytotoxicity or DNA degradation were apparent. However, above 145 nM of CAM, 900 nM of TN, or 2 microM of m-AMSA extensive DNA degradation in nuclei of S phase cells was evident within 6 h of addition of the inhibitor, resulting in the loss of S and G2 + M cells from these cultures. The data indicate that depending on concentration, mechanisms mediating the cytostatic/cytotoxic activity of both DNA topoisomerase I and II inhibitors may be quite different. Suppression of the DNA replication and the G2 to M transition, seen at low inhibitor concentrations, is compatible with the assumption that the inhibitor-induced stabilization of the topoisomerase-DNA cleavable complexes interferes with DNA replication and chromosome condensation/segregation, respectively. Above the threshold concentration for each inhibitor, an endonucleolytic activity is triggered, resulting in rapid DNA degradation in nuclei of S and G2 phase cells. The endonucleolytic effect is not only cell cycle phase-specific but is also modulated by tissue-specific factors because it cannot be observed, e.g., in the lymphocytic leukemic cell lines.     

Analgesic responses to intrathecal morphine in relation to CSF concentrations of morphine-3,beta-glucuronide and morphine-6,beta-glucuronide.

This study was performed to determine whether variations in analgesic responses to intrathecal morphine could be explained by cerebrospinal fluid (CSF) concentrations of morphine metabolites. Twenty-four CSF samples were collected at the beginning, middle and end of treatment periods in seven cancer patients with pain of malignant origin. CSF concentrations of morphine-3,beta-glucuronide (M3G) and morphine-6,beta-glucuronide (M6G) metabolites were measured by gas chromatography/mass spectrometry. Analgesic responses to morphine were estimated concurrent with CSF collection using a visual analog scale representing percentages of pain relief. Effective analgesia was defined as > or = 75% pain relief. CSF concentration of M3G and M6G in the 24 samples were 722 +/- 116 ng/ml and 699 +/- 158 ng/ml, respectively. CSF samples were categorized into two groups: (1) those collected during effective analgesia (N=14), and (2) those collected during ineffective analgesia (N=10). M6G levels detected in group 1 samples (effective analgesia) were significantly greater than those found in group 2 samples (ineffective analgesia) (978 +/- 243 ng/ml vs 309 +/- 68 ng/ml, P<0.05). Intergroup differences in CSF M3G concentrations and M3G/M6G ratios were not significant. It is concluded that CSF M6G may be indicative of effectiveness of analgesia in cancer patients subjected to intrathecal morphine.     

Evaluation of a method for detection of cells with reduced drug retention in solid tumours.

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra-assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter-assay reproducibility was too bad, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.     

Paclitaxel Induces Primary and Postmitotic G1 Arrest in Human Arterial Smooth Muscle Cells

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G1 and (b) post-mitotic arrest in G1. Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G1 arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G1 arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G1 but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg, -Trp, mePhe]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, -Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Raltegravir Cerebrospinal Fluid Concentrations in HIV-1 Infection

Introduction

Raltegravir is an HIV-1 integrase inhibitor currently used in treatment-experienced HIV-1-infected patients resistant to other drug classes. In order to assess its central nervous system penetration, we measured raltegravir concentrations in cerebrospinal fluid (CSF) and plasma in subjects receiving antiretroviral treatment regimens containing this drug.

Methods

Raltegravir concentrations were determined by liquid chromatography tandem mass spectrometry in 25 paired CSF and plasma samples from 16 HIV-1-infected individuals. The lower limit of quantitation was 2.0 ng/ml for CSF and 10 ng/ml for plasma.

Results

Twenty-four of the 25 CSF samples had detectable raltegravir concentrations with a median raltegravir concentration of 18.4 ng/ml (range, <2.0–126.0). The median plasma raltegravir concentration was 448 ng/ml (range, 37–5180). CSF raltegravir concentrations correlated with CSF:plasma albumin ratios and CSF albumin concentrations.

Conclusions

Approximately 50% of the CSF specimens exceeded the IC₉₅ levels reported to inhibit HIV-1 strains without resistance to integrase inhibitors. In addition to contributing to control of systemic HIV-1 infection, raltegravir achieves local inhibitory concentrations in CSF in most, but not all, patients. Blood-brain and blood-CSF barriers likely restrict drug entry, while enhanced permeability of these barriers enhances drug entry.     

Comparison of the effects of zinc deprivation and actinomycin D on ribonucleic acid synthesis by stimulated lymphocytes.

1. EDTA inhibited incorporation of [3H]uridine into RNA of lymphocytes, but did not decrease uptake into the cold-acid-soluble fraction of the cells. The inhibition by EDTA was largely reversible by simultaneous addition of Zn2+. 2. Low concentrations pf actinomycin D (3 ng/ml) added at the time of stimulation of the cells inhibited [3H]uridine incorporation into RNA, but concentrations of 50-100 ng/ml were required to produce the same degree of inhibition if addition of actinomycin D was delayed until just before the incorporation was measured. This difference in sensitivity did not reg within the cells. 3. When added immediately before phytohaemagglutinin, actinomycin D (3 ng/ml) and EDTA produced similar time-courses of inhibition of uridine incorporation. 4. Uridine incorporation at 32h was inhibited when actinomycin D (3 ng/ml) or EDTA was added just before stimulation of the cells, but was only slightly affected when they were added at 32h. At intermediate times the incorporation of uridine remained sensitive to addition of EDTA for longer than it was sensitive to actinomycin D. 5. Polyacrylamide-gel separation of RNA synthesized in EDTA-treated cultures in the presence or absence of added Zn2+ showed that lower availability of Zn2+ resulted in a decreased rate of transfer of radioactivity from 32S to 28S rRNA and decreased survival of 28S rRNA relative to 18S rRNA. 6. Close similarities have been shown to exist between the effects of EDTA and low concentrations of actinomycin D. Not all the effects of EDTA could be explained by postulating that Zn2+ was a constituent of RNA polymerase I, nor were the effects of actinomycin D readily explained by previously suggested mechanisms of action of this antibiotic.     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Exponential 3T3 cells escape in mid-G1 from their high serum requirement.

A simple, new method for determining the temporal location of arrests induced within the cell cycle is described. This method has the advantage that the initial, exponential cell population is unperturbed. It requires neither cell synchronization nor prior arrest of cells by starvation. The method involves partitioning cells located before and after the arrest point into classes of different DNA content. The magnitude of these classes, determined by flow microfluorimetry, is used to calculate the time of arrest within the cell cycle. The calculation utilizes an age distribution function which incorporates variability in cell-cycle durations. The method is used to derive the median time in the cell cycle when low serum arrests exponential Swiss 3T3 cells. The median durations of G1, S, G2 and M in these cells were: 5.4, 8.5, 3.0, and 0.7 h, respectively. Proliferating G1 cells with a median age of up to 3.2 h were blocked from entering S by reducing the exogenous serum concentration. G1 cells closer than approx. 2 h to S, S, G2 and M cells continued to transit the cell cycle. Preincubation of the cells in higher initial serum concentrations failed to alter this median age, indicating that adherence of serum factors to the cells does not influence the time determined. The data indicate that the G1 serum-sensitive events which finally direct cells toward either S or G0 are completed after approx. 2 h before S. Exposure to high serum apparently does not turn on DNA synthesis directly, but initiates an approx. 2 h sequence of required, late G1 events leading to S phase.     

Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography-mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5-200 ng/ml morphine and M6G, and 5-2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, d-Trp7,9, mePhe8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, d-Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Cytotoxicity and cytokinetic effects of mitomycin C and/or photochemotherapy in a human colon adenocarcinoma cell line.

1. The cytotoxicity and cytokinetic effects of Mitomycin C (MC) and/or photochemotherapy (PCT) in cultured human colon adenocarcinoma (WiDr) cells were investigated using colony formation to determine cell survival and DNA flow cytometry to analyze cell kinetics. 2. A low concentration of MC (0.01 micrograms/ml) caused accumulation of cells in late S and early G2 phase; higher concentrations (0.05-0.5 micrograms/ml) induced accumulation of the cells in mid and early S phase. 3. The effects of the lowest concentration of MC (0.01 micrograms/ml) were reversible upon removal of the drug, whereas a higher concentration of MC (0.1 micrograms/ml) resulted in a permanent inhibition of cell cycle progression. 4. The sensitivity of Photofrin II-loaded cells to PCT can be enhanced significantly by the addition of MC. 5. The MC-induced accumulation of the cells in S phase may be one reason for the increased cytotoxicity of PCT combined with MC. 6. The data suggest that MC may also inhibit repair of PCT-induced DNA damage.