S-phase modulation by irinotecan: pilot studies in advanced solid tumors.

Two studies of irinotecan (CPT-11) followed 24 h later by an antimetabolite were conducted. The objectives of the studies were: (1) to determine whether the increase in S-phase in tumor cells seen 24 h after CPT-11 administration in animal studies is seen in advanced solid tumors in patients, (2) to determine the dose of CPT-11 required to produce this effect, (3) to compare two methods (immunohistochemistry, IHC, for cyclin A, and DNA flow cytometry, FC) for evaluating S-phase in tumor biopsies from patients, and (4) to establish the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of CPT-11, given 24 h before gemcitabine (GEM, 1000 mg/m(2)). In one study CPT-11 was followed 24 h later by 5-fluorouracil (5-FU), 400 mg/m(2) per week for 4 weeks every 6 weeks. Tumor biopsies were obtained before and 24 h after CPT-11 administration before administration of 5-FU and assayed for S-phase by IHC for cyclin A and by FC. The starting dose of CPT-11 was 80 mg/m(2) per week with subsequent exploration of 40 and 60 mg/m(2) per week to establish the dose-effect relationship of the increase in tumor cells in S-phase. In the second study, CPT-11 was given 24 h before GEM 1000 mg/m(2) per week for 2 weeks every 3 weeks. Doses of 20-80 mg/m(2) were explored to establish the MTD and DLT and to study tumor cell S-phase in selected patients. CPT-11 80 mg/m(2) produced a mean increase in S-phase by IHC for cyclin A of 137%. Lesser increases were seen with 40 and 60 mg/m(2). CPT-11 followed 24 h later by 5-FU 400 mg/m(2) per week for 4 weeks was well tolerated. In the study of CPT-11 followed by GEM 1000 mg/m(2), 60 mg/m(2) of CPT-11 was the MTD.

Time-lapse video reveals immediate heterogeneity and heritable damage among human ileocecal carcinoma HCT-8 cells treated with raltitrexed (ZD1694).

BACKGROUND: Cellular heterogeneity in drug response has important clinical implications, and is believed to develop over many generations during clonal evolution in human tumors. The purpose of this study was to determine the level of heterogeneity exhibited by sister cells soon after their birth. METHODS: Human ileocecal carcinoma cells (HCT-8) were followed up to 11 days in vitro after a 2-h exposure to 1 microM raltitrexed (IC(95)) in a time-lapse video system. RESULTS: Over five experiments, 414 cells were followed after exposure to raltitrexed. Immediate sterility occurred in 74% of treated cells. Only 6% of cells could produce more than two generations of offspring, and heterogeneity in drug response was seen. Comparing sister cells < 24 h old, the more proliferative sibling produced up to 73 times more offspring, with a median ratio of 9.0 (control median = 1.19). Offspring of prolific drug-treated cells had a decreased probability of division (68% compared with 92%) and an increased average interdivision time (19.0 h compared with 15.1 h). CONCLUSIONS: Short-term exposure to raltitrexed resulted in increased interdivision times and production of sterile offspring extending seven generations. Cellular heterogeneity (difference in proliferation potential comparing drug-treated sister cells) was evident without a period of clonal evolution.

Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350.

Cellular topoisomerase I is an important target in cancer chemotherapy. A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A253 cells in vitro. As a basis for future clinical trials of BNP1350 in human head and neck carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by BNP1350 was correlated with different profiles of cell cycle-regulatory protein expression. When treated with the low concentration of BNP1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint kinase chk1 was observed when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage appears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induced alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.

Involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-associated spontaneous apoptosis and differentiation in an A253 human head and neck carcinoma xenograft model.

Time-dependent ladder-type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squamous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after transplantation, without any drug treatment. No evidence of ladder-type DNA fragmentation was detected in A253 cells in vitro or in normal nude mouse tissues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl-2 protein expression decreased, while bax protein expression increased from day 9 after transplantation. Moreover, altered expression of bcl-2 and bax was accompanied by the increased proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Time-dependent dephosphorylation of Rb, followed by proteolytic cleavage, was also observed from day 9 after transplantation. The data indicate that the caspase-3 activation and cleavage of Rb protein may represent important steps in the regulation pathway of bax-mediated spontaneous apoptosis. Interestingly, the time-dependent activation of spontaneous apoptosis was almost simultaneous with the induction of differentiation and increased expression of several differentiation-associated regulatory proteins. An increased expression of cyclin D1 and cyclin-dependent kinase-5 (cdk5) was observed from day 9 after transplantation, whereas only slight alteration of cdk4 expression was found. The time-dependent activation of cyclin D1 and cdk5 preceded both the induction of ladder-type DNA fragmentation and increased keratin pearl formation. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differentiation. Our observations suggest the involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-mediated spontaneous apoptosis and differentiation in A253 xenografts. P53 and WAF1 proteins were not expressed in the xenografts, indicating that the changes in the regulatory proteins during apoptosis and differentiation were not p53 or WAF1 dependent.

Parabolic growth patterns in 96-well plate cell growth experiments.

In preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1-5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.

Prevention of quinone-mediated DNA arylation by antioxidants.

High performance liquid chromatographic (HPLC) analysis showed that the prototype antioxidant ascorbate (vitamin C) inhibits the DNA adducts induced by synthetic estrogen diethylstilbestrol (DES) and the antiestrogen metabolite 4-hydroxytamoxifen (4-OHTam). Treatment of salmon testes DNA with 4-OHTam quinone or 4-OHTam in the presence of horseradish peroxidase and hydrogen peroxide (H(2)O(2)) generated the same DNA adduct profile. Vitamin C and N-acetylcysteine (NAC) inhibited the formation of 4-OHTam-dG adducts in a dose-dependent manner. To determine whether the same antioxidants also protect cellular DNA, HL-60 cells were used as cell culture model. Cells treated with 10 microM 4-OHTam in the presence of 1 microM H(2)O(2 )for 24 h gave 4-OHTam-dG adducts approximately 4 x 10(-7), n = 3. Treatment of the cells with 100 microM 4-OHTam, without H(2)O(2), produced the same level of adducts. Supplementation of the incubation media with vitamin C (2.5 mM) or NAC (5 mM) inhibited the formation of DNA adducts. Thus, antioxidants may protect susceptible cells from genotoxicity associated with 4-OHTam activation.

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin -6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034): comparison across sensitive and resistant human tumor cell lines.

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

Persistent induction of apoptosis and suppression of mitosis as the basis for curative therapy with S-1, an oral 5-fluorouracil prodrug in a colorectal tumor model.

In an effort to improve the therapeutic selectivity of 5-fluorouracil (FUra) against colorectal cancer, S-1, a combination agent including a prodrug of FUra with two modulators, was recently developed by Taiho Pharmaceuticals Co. S-1 is a combination of tegafur (FT), 5-chloro-2,4-hydroxypyridine, and potassium oxonate in the molar ratio of 1.0:0.4:1.0, with the latter two components as inhibitors of dihydropyrimidine dehydrogenase and phosphoribosylpyrophosphate transferase, respectively. In this study, the therapeutic selectivity and efficacy of S-1 (oral) was compared with FT (oral) and FUra (i.v. infusion) in rats bearing advanced colorectal cancer by using clinically relevant schedules. The maximum tolerated doses (MTDs) of S-1, FT, and FUra were 31.5, 200, and 25 mg/kg/d for 7 days and 22.5, 150, and 12.5 mg/kg/d for 28 days, respectively. The therapeutic index of S-1 was 4- to 5-fold higher than that of either FT or FUra. S-1 achieved 100% complete tumor regression (CR) at its MTD in both 7-day and 28-day schedules. Furthermore, the high incidences of stomatitis, alopecia, and diarrhea observed with FUra and FT, were not observed with S-1. In an attempt to understand the basis for the observed superior therapeutic selectivity with S-1, we studied pharmacokinetic analysis of FUra, drug-induced apoptosis, suppression of mitosis, and inhibition of thymidylate synthase (TS) after S-1, FUra, or FT administration. The peak plasma FUra concentrations derived from FUra or S-1 (FT) at comparable MTDs were similar, but the plasma level of FUra was higher with S-1 than with FUra. Induction of high and sustained apoptosis was achieved with S-1. Although the initial level of apoptosis induced by FUra was comparable to S-1, it was not sustained. The sustained level of apoptosis appears to correlate with tumor growth inhibition. Mitotic figures were more greatly suppressed with S-1 treatment than with FUra. Studies on TS inhibition indicated that, although both S-1 and FUra caused a 4- to 6-fold induction of total TS protein, single oral administration of S-1 was superior to 24-h infusion of FUra in suppressing free TS. The data are consistent with the observation that the therapeutic efficacy of S-1 (100% cure) over FUra is associated with high and sustained levels of drug-induced apoptosis, greater suppression of mitosis, and inhibition of free TS in tumor tissues.

Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex.

Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.

Image analysis for quantitation of solid tumor drug sensitivity.

BACKGROUND: A method of assessing chemosensitivity of tissue has been described by Rotman et al. The aim of this study was to use image analysis to provide a more rapid and quantitative means of assessing drug effect on tissue proliferative capacity. METHOD: Fluoropyrimidine sensitive Ward rat colon adenocarcinoma tumor was implanted onto collagen impregnated cellulose fibers suspended on metal grids at an air-fluid interface and kept in a 95% air/5% CO2 incubator at 37 degrees C. The fluorescent microscopic image captured by a silicon intensified target (low light detecting) camera and linked to an image processing unit was measured for fluorescent brightness and tumor image area. Blinded 5-Fluorouracil (5-FU) drug treatment was begun 8 days after tumor explantation on the collagen-cellulose matrix. Tumor image area and fluorescent brightness were measured at 24 h pretreatment, 48 h posttreatment, and at 48 h post drug removal. RESULTS: Nontreated tumor cultures demonstrated an increase in area and fluorescent brightness with time following tumor implantation on the collagen gel. Dose responsiveness was seen with increasing concentrations of 5-FU. At the highest clinically achievable concentration of 5-FU (500 microM), there was a 39% decrease in area compared with the nontreated group, 113%. Linear dose responsiveness was not demonstrated between 50 and 150microM 5-FU. CONCLUSIONS: Fluoropyrimidine activity was demonstrated with the implemented image analysis system. The in vitro tumor sensitivity to FU using collagen gel was consistent with responsiveness of tumors in vivo borne by rats.

Modeling of the time-dependency of in vitro drug cytotoxicity and resistance.

For potential clinical extrapolation of in vitro findings, it is of interest to relate the measured effect of an anticancer agent to concentration and exposure time. The Hill model (A. V. Hill, J. Physiol., 40: iv-vii, 1910) is commonly used to describe pharmacodynamic (PD) effects, including drug-induced growth inhibition of cancer cells in vitro. The IC(X)n x T = k relationship, in which IC(X) is the concentration of agent required to reduce cell growth by X%, T is the exposure time, and n and k are estimable parameters, was first applied to bacterial disinfectant action and then was successfully used to model anticancer drug potency as a function of exposure time (D. J. Adams, Cancer Res., 49: 6615-6620, 1989). Our goal was to create a new global PD modeling paradigm to facilitate the quantitative assessment of the growth-inhibitory effect of anticancer agents as a function of concentration and exposure time. Wild-type human ovarian A2780 and ileocecal HCT-8 carcinoma cells and sublines that were resistant to cisplatin (A2780/CP3), doxorubicin (A2780/DX5B), and raltitrexed (RTX) (HCT-8/DW2) were exposed to various anticancer agents, cisplatin, doxorubicin, paclitaxel, trimetrexate, RTX, methotrexate, and AG2034, for periods ranging from 1 to 96 h. Cell growth inhibition was measured with the sulforhodamine B protein dye assay. Patterns of time-dependency of drug potency, slope of the concentration-effect curves, and relative degree of resistance were characterized. Empirical mathematical expressions were built into a global concentration-time-effect model. The global PD model was then fit to the concentration-time-effect data with iteratively reweighted nonlinear regression. Under specific treatment conditions, the examination of the slope and the shape of the concentration-effect curves revealed a large heterogeneity in drug response, e.g., shallow concentration-effect curve or double or triple Hill "roller coaster" concentration-effect curve. These patterns, which were observed at intermediate exposure times in parental and resistant cells for paclitaxel and trimetrexate or only in resistant HCT-8/DW2 cells for RTX, methotrexate, and AG2034, revealed mechanistic insights for the former cases but possible methodological artifacts for the latter cases. The comprehensive PD modeling of the cytotoxic effect of anticancer agents showed that it was possible to modulate drug effect, response heterogeneity, and drug resistance by altering the time of exposure to the agents. This approach will be useful for: (a) describing complex concentration-time-effect surfaces; (b) refining biological interpretations of data; (c) providing insights on mechanisms of drug action and resistance; and (d) generating leads for clinical use of anticancer drugs.

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation.

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.

HsMCM2/BM28: a novel proliferation marker for human tumors and normal tissues.

HsMCM2/BM28 is a member of the family of minichromosome maintenance (MCM) proteins, which play a critical role in DNA replication by helping to ensure that DNA is replicated once and only once per cell cycle. The association of HsMCM2 with DNA replication suggested to us that it might prove useful as a new marker for cell proliferation. To test this possibility, we employed immunohistochemistry and immunoblotting to study HsMCM2 expression in both normal human tissues and primary human tumors. We found that HsMCM2 was detectable by immunoblotting in 97% of the studied tumors but in only 27% of the corresponding normal tissues. In normal tissues, the immunoblot signal was weaker than in tumors. Immunohistochemistry revealed that in normal tissues HsMCM2 is present only in proliferating cell nuclei. In most cases, tumor cell nuclei produced a stronger HsMCM2 signal than normal proliferating cell nuclei. Comparative studies revealed that antibodies against HsMCM2 stained fewer nuclei than antibodies against proliferating cell nuclear antigen but usually more than antibodies against Ki-67 (another proliferation-related antigen). Thus, the correlations between proliferation and antigenic signal are different for these three proteins. These results indicate that HsMCM2 is, indeed, a novel marker for proliferating cells. Further studies are required to determine whether the fact that HsMCM2 has a different correlation with proliferation and elevated staining intensity in tumor nuclei (compared to nuclei in normal proliferating cells) will permit it to be a more useful diagnostic and prognostic marker than proliferating cell nuclear antigen and Ki-67.

Implications for clinical pharmacodynamic studies of the statistical characterization of an in vitro antiproliferation assay.

Modeling of nonlinear pharmacodynamic (PD) relationships necessitates the utilization of a weighting function in order to compensate for the heteroscedasticity. The structure of the variance was studied for concentration-effect data generated in an in vitro 96-well plate cell growth inhibition assay, where data are numerous (480 data points per experiment) and replication is easy. From the five candidate models that were considered, the power function S2Y = phi 2Y phi 3, where Y is the sample mean and S2Y is the sample variance, was shown to be the most appropriate to describe the nonuniformity of the variance along the range of measured effect for 253 sets of (Y; S2Y) data. The Hill model was fit to the concentration-effect data with weighted nonlinear regression, where the weights were equal to the reciprocal of the predicted variance. The examination of the distribution of the 253 sets of parameters of the PD model showed that IC50 was lognormally distributed whereas the distribution of gamma was normal. The characterization of the appropriate variance function and concentration-effect function in a simple in vitro experimental setting with a large number of experiments, with each experiment including a large number of data points, will be useful for guiding similar in vitro concentration-effect studies where data are plentiful and for guiding PD modeling in complex clinical settings in which extensive data for model characterization is impossible to obtain.

Mechanisms of acquired resistance to modulation of 5-fluorouracil by leucovorin in HCT-8 human ileocecal carcinoma cells.

Repeated (10x) exposure of HCT-8 human ileocecal carcinoma cells to 5-fluorouracil (5-FU) for 2 or 72 hr, both incubations in the continuous presence of 20 microM leucovorin (LV), yielded two stable modulation-resistant sublines, FL2h and FL72h. Although LV potentiated growth inhibition by 5-FU 2-fold in parental HCT-8 cells, it did not potentiate the effect of 5-FU in the FL2h or FL72h sublines. LV modulation of 5-fluorodeoxyuridine (5-FdUrd) was also reduced (FL72h) or eliminated (FL2h). In the FL2h and FL72h sublines, the level of thymidylate synthase (TS) protein and TS activity in cell extracts, TS activity in situ, the rate of cellular uptake and metabolism of LV, and the level of 5-FU incorporation into total cellular RNA were similar to those in parental HCT-8 cells. However, LV significantly (P < 0.01) potentiated the inhibition of TS activity in situ in HCT-8 cells at 24 hr after a 2-hr treatment with either 5-FU or 5-FdUrd, but had no such activity in the FL2h and FL72h sublines (P > 0.1). Resistance to modulation of 5-FU by LV was associated with the inability of LV to increase the formation of intracellular TS-FdUMP-methylenetetrahydrofolate ternary complexes, and these complexes dissociated more rapidly (T1/2 > 1.5- to 3-fold faster) in the presence of different concentrations of 5,10-methylenetetrahydropteroylpentaglutamate. Thus, decreased stability of ternary complexes appears to be the mechanism of acquired resistance to the LV modulation of fluoropyrimidine cytotoxicity, possibly due to mutation(s) of TS in these two modulation-resistant HCT-8 sublines.

Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells.

PURPOSE: The combination of paclitaxel (PTX) and cisplatin (DDP) shows good clinical efficacy against ovarian cancer. In order to examine the potential cellular basis for this, and provide leads as to how to optimize the combination, we examined the role of sequence of exposure to PTX and DDP on cell growth in vitro. METHODS: Four human ovarian carcinoma cell lines, A121, A2780/WT, A2780/DX5B and A2780/CP3, two human head and neck carcinoma cell lines, A253 and FaDu, and the human ileocecal carcinoma cell line, HCT-8, were treated with PTX + DDP with seven schedules: (A) 96 h exposure to PTX + DDP; (B) 24 h PTX alone, then 72 h PTX + DDP; (C) 4 h DDP alone, then 92 h PTX + DDP; (D) 24 h PTX alone, 4 h DDP alone, then 68 h drug-free; (E) 4 h DDP alone, 24 h PTX alone, then 68 h drug-free; (F) 3 h PTX alone, 1 h DDP alone, then 92 h drug-free; and (G) 1 h DDP alone, 3 h PTX alone, then 92 h drug-free. Each of 66 two-drug experiments included five plates (440 randomly treated wells per experiment). Cell growth was measured by the sulforhodamine B assay. The nature and the intensity of the drug interactions were assessed by fitting a seven-parameter model to data with weighted nonlinear regression, enabling the estimation of an interaction parameter, alpha, with its standard error. RESULTS: Overall there was very little departure from Loewe additivity: 43 experiments showed Loewe additivity, 10 showed Loewe antagonism, and 13 showed slight Loewe synergy. In vitro Loewe synergy was rare, was small when present, and reproducible only for the A121 and HCT-8 cells exposed to schedule D (24 h PTX prior to 4 h DDP). Isobolographic analysis showed complex combined-action surfaces with regions of local Loewe synergy and antagonism. CONCLUSION: It appears unlikely that the good clinical efficacy of the combination is primarily caused by a synergistic interaction at the cellular level.

MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumors.

The expression of human MDR1 P-glycoprotein (Pgp) in the capillary endothelial cells of the central nervous system has been demonstrated. The brain capillary endothelial cells maintain the structure and function of the blood-brain barrier. Recently, the human MDR1 Pgp (and its mouse homologue MDR1a Pgp) has been shown to function as an important part of this barrier, pumping out xenobiotics from endothelial cells into the lumen of capillaries resulting in the protection of the brain parenchyma. To examine whether the endothelial cells of the newly formed capillaries during neoangiogenesis within malignant human brain tumors express MDR1 Pgp, 35 adult surgical brain tumor specimens (29 gliomas and 6 tumors metastatic to the brain) were obtained from previously untreated patients and studied by a new immunohistochemical sandwich method developed in our laboratory using the JSB-1 monoclonal antibody. JSB-1 is specific for the Pgp product of the human MDR1 (and not MDR3) gene. This sensitive method allows the detection of Pgp in capillary endothelial cells of normal brain in conventional paraffin sections after formalin fixation. The endothelial cells of the newly formed capillaries in 25 of 29 gliomas (86%) and 3 of 6 metastatic tumors, immunostained positive for MDR1 Pgp. The tumor cells in 7 of 35 cases were also positive for Pgp. In the 35 brain tumor cases investigated, the endothelial cells were Pgp positive in the tumor-brain border and in the brain further from the tumor. Capillary endothelial cells of neovasculature in 137 malignant tumors (non-brain) obtained from previously untreated patients showed no MDR1 Pgp expression. These results demonstrated that MDR1 Pgp is expressed not only in the capillaries of normal brain but also in the majority of the newly formed capillaries of brain tumors. Multidrug resistance of brain tumors may result not only from the expression of resistance markers in neoplastic cells but also from the MDR1 Pgp expression in endothelial cells of tumor capillaries. Pgp in this special localization can exclude chemotherapeutic agents from tumor cells that are located around the capillaries. The therapeutic benefit and selectivity of chemotherapeutic agents in combination with a Pgp-reversing agent should be evaluated.

Capillary electrophoretic separation and laser-induced fluorescence detection of the major DNA adducts of cisplatin and carboplatin.

Micellar electrokinetic capillary chromatographic separation of a dansylated mixture of normal nucleotides and platinated cross-link adducts of d(pGpG) and d(pApG) was optimized with baseline resolution. The introduction of a laser-induced fluorescence (LIF) detector overcame the lack of sensitivity characteristic of capillary electrophoresis (CE) due to the small injection volume and the short optical path length. CE/LIF was able to detect 1 adduct/10(4) normal nucleotides/micrograms DNA by fluorescence postlabeling assay. The enrichment of the adduct, prior to dansylation, enhanced the detection limit to 1 adduct/10(7) normal nucleotides/micrograms DNA. Calf thymus DNA was reacted in vitro with cisplatin and carboplatin with total input drug/nucleotide ratios of 0.05 and 0.5, respectively. A2780 human ovarian carcinoma cells were exposed in culture to 25 microM cisplatin for 2 h. The cells were incubated with drug-free medium for 3 h before harvesting. The identification of the cross-link adducts in modified DNA was confirmed by cochromatography with authentic markers. The same 1,2-intrastrand cross-link adducts were induced by both cisplatin and its second-generation drug carboplatin. This report has demonstrated, for the first time, the utility of CE/LIF as an analytical tool for assaying DNA damage [corrected].

Cellular heterogeneity in DNA damage and growth inhibition induced by ICI D1694, thymidylate synthase inhibitor, using single cell assays.

Heterogeneity in the response of the HCT-8 (human ileocecal adenocarcinoma) tumor cell line to a new thymidylate synthase inhibitor, ICI D1694, was investigated in terms of induction of DNA single-strand breaks and cytotoxicity, applying the single cell alkaline gel (SCG) electrophoresis assay and the individual colony formation assay (iCFA), respectively. ICI D1694 induced maximal total DNA single-strand breaks 24 hr after a 2-hr drug exposure with incomplete repair by 72 hr. The level of DNA damage was concentration dependent and paralleled cellular growth inhibition in vitro. The proportion of cells with DNA damage and the extent of DNA single-strand breaks increased with drug concentration. At 1 microM ICI D1694 (IC95), a significant level of DNA damage was detected in 58% of the cells; however, 25% of the cells had little or no damage. Using the iCFA system, it was observed that with 1 microM ICI D1694, only 2.6% of the seeded cells maintained a colony growth rate similar to that of the control colonies, and 22% of the cells were growing significantly more slowly. In conclusion, the SCG assay and the iCFA identified subpopulations of cells that were unaffected by ICI D1694. Although these cells represented only a small proportion of the total cell population, this phenomenon of heterogeneity in response to ICI D1694 might limit its therapeutic efficacy.

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.