Biomedical subjects

G J Finlay

Publications and source records attributed to G J Finlay.

At least 55 records · Page 3Linked to original sources

PubMed1992

Microculture-based chemosensitivity testing: a feasibility study comparing freshly explanted human melanoma cells with human melanoma cell lines.

BACKGROUND: The culture of cancer cells has many applications in chemosensitivity testing and new drug development. PURPOSE: Our goal was to adapt simple semiautomated microculture methods for testing the chemosensitivity of melanoma cells freshly recovered from patients' tumors. METHODS: Cells were cultured on a substrate of agarose and exposed continuously to cytotoxic drugs, the effects of which were measured by determining the uptake of [3H]thymidine 4-7 days later. RESULTS: Immunocytochemical staining of cells cultured with 5-bromo-2'-deoxyuridine demonstrated that tumor cells were responsible for the measured thymidine incorporation. The effects of cytotoxic drugs were calculated as logarithmic 50% inhibitory concentrations and expressed as divergences from the mean in a log-mean graph. The inhibitory effects of amsacrine, etoposide, doxorubicin, cisplatin, mitomycin C, and fluorouracil were tested. Tumors differed widely in their sensitivity to these drugs, although sensitivity to the three topoisomerase-II-directed agents was highly correlated. Cells from two non-neoplastic hematopoietic progenitor cell lines (FT and 32D) showed chemosensitivity patterns distinct from those in the melanoma cells, indicating tissue selectivity. Two established melanoma cell lines, MM-96 and FME, were tested under the same conditions and showed sensitivity typical of at least some fresh specimens. CONCLUSIONS: These results support the validity of melanoma cell lines as models of freshly resected melanoma cells. If successfully applied to other tumor types, such semiautomated approaches could find wide application in routine hospital laboratories for the chemosensitivity testing of patients' tumor cells.

Animals↗

PubMed1992

Melphalan penetration of the blood-brain barrier via the neutral amino acid transporter in tumor-bearing brain.

Melphalan, a nitrogen mustard derivative of the neutral amino acid L-phenylalanine, was transported across the rat blood-brain barrier by the large (L-system) neutral amino acid transporter in tumor-bearing brain, but no evidence for blood-brain barrier transport by the alanine-serine-cysteine system carrier was obtained in the present study. The ability of melphalan to inhibit phenylalanine uptake was compared in rats implanted with two experimental CNS tumors: the C-6 glioma (a model of primary brain tumors) and Walker carcinoma (a model of metastatic brain tumors). The melphalan concentration which caused 50% inhibition of blood-brain barrier (BBB) phenylalanine uptake (Ki) was 0.49 +/- 0.18 mM in the Walker tumor, compared with 0.46 +/- 0.19 mM in the contralateral control brain. In the ipsilateral hemisphere (Ki = 0.59 +/- 0.25 mM) and contralateral hemisphere (Ki = 0.45 +/- 0.19 mM), drug entry was also via the neutral amino acid transporter. In C-6 gliomas (Ki = 0.77 +/- 0.20 mM) and contralateral control brain (Ki = 0.84 +/- 0.29 mM), melphalan also inhibited BBB phenylalanine transport. A major finding was that, at melphalan concentrations greater than 1.0 mM, BBB permeability of radiolabeled indium (chelated to EDTA) increased in proportion to melphalan concentration. In the contralateral hemisphere of rats implanted with C-6 gliomas, brain extractions of indium-EDTA measured 3 to 4% in the absence of drug, 5 to 6% at 2.5 mM melphalan, and 9 to 10% at 5 mM melphalan. A similar phenomenon was observed in the nontumoral brain regions of rats implanted with Walker carcinoma cells. In normal (nonimplanted) rats, melphalan's inhibition (Ki = 0.29 mM) of phenylalanine and tryptophan (Ki = 0.20 mM) uptake was confirmed, and brain extraction of sucrose (a nonspecific marker which does not penetrate the intact BBB) was observed to increase in proportion to melphalan concentration. We conclude that melphalan not only enters the brain via the neutral amino acid transporter, but at higher concentrations (greater than 1 mM) may open the blood-brain barrier in a nonspecific manner.

Amino Acid Transport Systems↗

PubMed1992

Unusual dynamics of killing of cultured Lewis lung cells by the DNA-intercalating antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide.

The cytotoxicity of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601,316), a new experimental DNA-intercalating antitumor drug, against a cultured Lewis lung adenocarcinoma cell line was compared with that of the DNA-intercalating antitumor drug amsacrine. In contrast to amsacrine, AC demonstrated self-inhibition of cytotoxicity following short (3-9 h) incubation periods and exponential killing (with a shoulder) after long (24-72 h) periods of incubation. The difference between these drugs was best demonstrated using a constant concentration x time (C x T) exposure (AC, 12 mumol h l-1; amsacrine, 3 mumol h l-1). In contrast to amsacrine, AC was minimally effective over exposure periods of less than or equal to 1 h and maximally effective over intermediate periods (4-6 h). The results suggest the possibility of designing AC administration protocols that maximise the drug's cytotoxicity towards solid tumors, which, because of diffusion barriers, are subjected to longer drug exposures than are well-vascularised tumours.

Acridines↗

PubMed1992

Relationship of cell cycle parameters to in vitro and in vivo chemosensitivity for a series of Lewis lung carcinoma lines.

The sensitivity of three Lewis lung carcinoma sublines, which grow in culture and in vivo, and vary in in vivo drug sensitivity, have been compared using topoisomerase II poisons amsacrine, amsacrine analogue CI-921, doxorubicin and etoposide. D10 (drug concentration for 10% clonogenic survival) values were determined in vitro for low and high density cultures, and ex vivo for cells from subcutaneous tumours. The cytokinetic parameters of these populations were obtained by flow cytometric analysis of bromodeoxyuridine-labelled cells. Regression analysis showed that logarithmic D10 values were significantly correlated (r greater than 0.95) with G1- and S-phase proportions and highly correlated (r = 0.99) with calculated G1 transit times. The slopes of the regression lines were similar for all topoisomerase II poisons tested and it is suggested that this slope reflects the disappearance of topoisomerase II during G1 phase.

Antineoplastic Agents↗

PubMed1992

Resistance mechanisms to topoisomerase poisons: the application of cell culture methods.

A survey is presented of two types of resistance to drugs acting on cellular topoisomerase enzymes, that occurring by altered drug transport and that occurring by altered target interaction. Particular attention is paid to the use of pairs of diagnostic drugs, one susceptible and one refractory to a particular resistance mechanism, in the determination of such resistance in cultured cells. Two pairs of drugs, each including the antitumor agent amsacrine, are used in the analysis of a number of cell lines. Also it is suggested that some normal hemopoietic precursor cells may exhibit two types of multidrug resistance, and that this should be taken into account in determining selectivity of topoisomerase-directed drugs for tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

In vitro methods for screening agents with an indirect mechanism of antitumour activity: xanthenone analogues of flavone acetic acid.

Xanthenone-4-acetic acid (XAA) resembles flavone acetic acid (FAA) in its effects on solid tumours in mice. The activity of methyl-substituted XAA derivatives in vitro was determined using 18 h 51Cr-release assays, continuous exposure growth inhibition assays and stimulation of tumouricidal activity of cultured murine resident peritoneal macrophages. The macrophage assay identified the high biological activity and dose potency of 5-MeXAA in vivo, and was the most accurate in vitro predictor of the ability of congeners to induce either haemorrhagic necrosis of subcutaneous Lewis lung and colon 38 tumours or splenic natural killer activity. In vitro immune stimulation may be more appropriate than direct cytotoxicity for screening compounds with indirect mechanisms of antitumour activity.

Animals↗

PubMed1990

Multiple patterns of resistance of human leukemia cell sublines to amsacrine analogues.

The antileukemia drug amsacrine and seven analogues were tested for in vitro activity against five multidrug-resistant human leukemia cell sublines (two derived from each of two Jurkat parent lines and one from the K562 line) and the corresponding parent lines (Jurkat, K562, and P388 leukemia). Resistant cell lines were derived by exposure to either amsacrine or doxorubicin. The resistance factor was calculated as the ratio of the mean IC50 value for the resistant cell line to that for the parent line. IC50 was defined as the concentration of drug inhibiting cell growth to 50% of that in control (drug-free) cultures. Patterns of cross-resistance were visualized by plotting the deviations of resistance factors from the mean resistance factor, on a logarithmic scale. Considerable variations in resistance factors were noted for each cell subline as the amsacrine substituents were altered. Four main patterns were evident: a transport-related multidrug-resistance pattern (three sublines), a pattern similar to that for a murine P388 leukemia subline resistant to amsacrine, and two patterns not observed previously. Since some of the sublines tested showed evidence of altered topoisomerase II activity, it appears that changes in the resistance pattern in these lines may reflect changes in the stability of the ternary complexes formed by the drug, the altered enzyme, and the DNA. The resistance factor was reduced from more than 100-fold to about 13-fold in three of the sublines tested and from eightfold to twofold in two others. This finding suggests that appropriate drug design may overcome several forms of multidrug resistance in human leukemia and other types of cancer.

Amsacrine↗

PubMed1990

Potentiation by phenylbisbenzimidazoles of cytotoxicity of anticancer drugs directed against topoisomerase II.

Analogues of the phenylbisbenzimidazole dye pibenzimol bind tightly to the minor groove of DNA. A clonogenic assay has been used to investigate the effects of these compounds on the cytotoxicity of the topoisomerase II directed anti-cancer drugs amsacrine, CI-921 (an amsacrine analogue), acridine carboxamide, etoposide and doxorubicin. Although pibenzimol itself was inactive, several of its analogues reduced the toxicity of etoposide, amsacrine and CI-921 towards a Lewis lung mouse tumour line at concentrations between 1 and 20 mumol/l. Doxorubicin cytotoxicity was unaffected, suggesting that this drug has a distinct mechanism of action. At concentrations below 1 mumol/l, some of these dyes potentiated the cytotoxicity of etoposide and CI-921 towards Lewis lung cells. Potentiation of CI-921 activity was also found with the human tumour lines HT29 (colon), SW620 (colon) and FME (melanoma). Novel treatments may arise from the potentiation of topoisomerase II directed cytotoxicity.

Acridines↗

PubMed1990

Comparison of the cytotoxicity of amsacrine and its analogue CI-921 against cultured human and mouse bone marrow tumour cells.

Human and mouse bone marrow cells were cultured for 1 h in the presence of either the antileukaemia drug amsacrine or its 4-methyl,5-[N-methyl]carboxamide disubstituted analogue CI-921, before being plated in methylcellulose medium to determine the survival of granulocyte-macrophage colony forming units (CFU-GM). The drug concentration required for 50% reduction in survival was approx. 0.4 microM for both drugs and was similar for both human and mouse cells. A comparison of the two drugs was then made, at an added drug concentration of 0.5 microM, using cultured mouse L1210 and P388 leukaemia, Lewis lung carcinoma cell lines LLAK and LLTC, human Jurkat leukaemia, human histiocytic lymphoma U937 and human colon carcinoma SW620. The sensitivity of the mouse lines for amsacrine was in the order L1210 greater than P388 greater than LLAK greater than LLTC, similar to the in vivo sensitivity. The selectivity of CI-921 for L1210 versus bone marrow, and for LLAK versus L1210 or P388, was greater than that of amsacrine, again in keeping with its in vivo properties. The sensitivity of the human Jurkat and U937 lines for amsacrine was intermediate between that of L1210 and P388, while SW620 was resistant. The selectivity of CI-921 for Jurkat and U937 versus bone marrow was greater than that of amsacrine, suggesting that CI-921 could have additional advantages over amsacrine in the treatment of some tumours.

Amsacrine↗

PubMed1989

Selectivity of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide towards Lewis lung carcinoma and human tumour cell lines in vitro.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601316) is a chemically novel antitumour agent which is thought to interact with DNA topoisomerase II and which has DNA binding properties which are distinct from other acridine derivatives such as amsacrine and its disubstituted analogue CI-921. AC is one of the most active agents, experimental or clinical, against the Lewis lung carcinoma in mice. AC is the first acridine derivative in our hands to show higher activity against cultured Lewis lung cells than against leukaemia lines. AC is more active against two human leukaemia cell lines (U-937 and Jurkat) than against a melanoma line (MM-96) and is inactive against the HT-29 human colon line. With all cell lines tested, cytotoxicity was higher at AC concentrations of 3-6 microM than at 15-20 microM. AC at a concentration of 20 microM inhibited the cytotoxicity of amsacrine and CI-921, but not that of another topoisomerase-directed drug doxorubicin. A Lewis lung line which had been cultured for a long period was less sensitive than a line freshly isolated from mice, but sensitivity of the cultured line recovered after it was multiply passaged in vivo. Long-term cultures may therefore be less appropriate than short-term cultures for predicting effectiveness of AC in vivo.

Acridines↗

PubMed1989

Chemoprotection by 9-aminoacridine derivatives against the cytotoxicity of topoisomerase II-directed drugs.

The effect of three acridine derivatives, 9-aminoacridine (9AA), 4'-(9-acridinylamino)-methanesulphon-O-anisidide (O-AMSA) and quinacrine were compared in their ability to protect against the cytotoxicity of amsacrine, 9-[[2-methoxy-4-[(methylsulfonyl)amino]phenyl]amino)-N,5-dimethyl-4- acridine-carboxamide (CI-921), N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC), etoposide, mitoxantrone and doxorubicin. Cytotoxicity was measured in vitro by clonogenic survival assay and in vivo by life extension assays. All three acridine derivatives protected a Lewis lung cell line in vitro against CI-921, with 9AA having the highest activity. Cellular uptake of [14C] CI-921 by cultured Lewis lung cells was unaffected by 9AA, and slightly stimulated by O-AMSA and quinacrine. 9AA protected Lewis lung cells in vitro against the cytotoxicity of amsacrine, CI-921, AC and etoposide, partially against mitoxantrone but not against doxorubicin. A similar result was obtained with the human melanoma cell line MM96, where 9AA protected against CI-921 but not against doxorubicin toxicity. 9AA protected P388 leukaemia in vivo against amsacrine, CI-921 and AC cytotoxicity, partially against etoposide but not against mitoxantrone or doxorubicin. 9AA also protected against animal toxicity caused by high dose amsacrine and partially against CI-921 toxicity. It is hypothesized that DNA intercalating chemoprotectors act by restricting the conformational flexibility of the DNA and thus the ability of topoisomerase II to form a 'cleavable complex' in which the DNA is covalently linked to the enzyme.

Aminacrine↗

PubMed1988

Effect of flavone acetic acid on Lewis lung carcinoma: evidence for an indirect effect.

Flavone-8-acetic acid (FAA), a new antitumor agent currently undergoing clinical trial, fails to inhibit the growth of early stage Lewis lung (LL) tumors growing in the lung. However, the growth of advanced subcutaneous tumors, arising from inoculation of either the original in vivo LL line or a tissue culture-adapted cell line (LLTC) derived from the LL line was delayed significantly by FAA treatment. Comparison, by clonogenic survival assays, of the cytotoxic effect of FAA on LLTC cells demonstrated that most cell killing occurred between 2 and 8 hours following in vivo exposure but occurred to a much lesser extent and at later times following in vitro exposure. FAA was inactive against LLTC cells growing in vivo in diffusion chambers, suggesting that a host cellular component was necessary for activity. FAA was found to induce hemorrhagic necrosis in the advanced LL tumors, as well as in a number of human tumor xenografts growing in athymic mice. The human cell lines from which the xenografts were derived, as well as the LL tumor lines and P388 leukemia lines, were inhibited by FAA in vitro. However, the ranking of FAA activity in vivo did not parallel that observed in vitro. Together, these observations strongly suggest that FAA has an indirect mode of antitumor action.

Animals↗

PubMed1988

Derivatives of amsacrine: determinants required for high activity against Lewis lung carcinoma.

Antitumor activity against the Lewis lung carcinoma in mice is reported for the series of 36 acridine-substituted derivatives of the antileukemia agent amsacrine. This series is the one from which the analogue N,5-dimethyl-9-[(2-methoxysulfonylamino)phenylamino]-4-acridinecarboxamide (CI-921), presently in clinical trial, was chosen. The analogues also were tested in vitro by comparing growth inhibition data [IC50 values (concentration required to reduce growth of cultured cells to 50% of that of untreated cultures)], using L1210 murine leukemia cells and HCT-8 human colon carcinoma cells. Determined IC50 values were highly dependent on the culture medium used, and it was found that the presence of ascorbate in the medium had a major effect on the stability of compounds to oxidation. A survey of 115 analogues of amsacrine indicates that a low ratio of IC50 values (HCT-8/L1210) is necessary but not sufficient for good antitumor activity against the solid tumor. DNA binding constants did not in themselves predict activity, although they were related to dose potency. Other factors, such as drug lipophilicity, acridine base strength, and drug solubility, also are involved, probably in providing effective drug distribution. It is concluded that in vitro assay data provide information useful for drug design but that other factors also are important for in vivo activity.

Amsacrine↗

PubMed1988

Relationship between the structure of analogues of amsacrine and their degree of cross-resistance to adriamycin-resistant P388 leukaemia cells.

A series of derivatives of 9-anilinoacridine related to the anti-leukaemia agent amsacrine have been tested in continuous exposure growth inhibition assays to determine the degree of cross-resistance in the Adriamycin-resistant P/ADR murine leukaemia line. Measured IC50 values for the two cell lines were only poorly correlated (r = 0.51), and cross-resistance as measured by the ratio of IC50 values varied from 2-fold and 272-fold. A high degree of resistance was found to be associated with the presence of amino or substituted amino groups on the acridine ring system. Logarithmic IC50 values were determined for other cell lines (L1210 leukaemia, Lewis lung carcinoma and HCT-8 human colon carcinoma) and were compared with those for the P388 lines to determine the degree of linear correlation. HCT-8 values were strongly correlated with P/ADR values (r = 0.84) while L1210 values correlated strongly with those of the sensitive P388 line (r = 0.98). Values for Lewis lung cells showed an intermediate pattern and correlated with a linear combination of values for both P388 lines (r = 0.88). Examination of available IC50 values for a number of rodent and human cell lines indicates that their sensitivity patterns are either P388-like or else intermediate between P388 and P/ADR. The series of amsacrine derivatives may be useful in characterizing the nature and degree of multidrug-resistance in cultured cell lines.

Amsacrine↗

PubMed1987

Abnormal insulin-receptor down regulation and dissociation of down regulation from insulin biological action in cultured human tumor cells.

The sensitivity of insulin receptors to down regulation by insulin has been measured in cultured human tumor cells (breast tumor cell lines MCF-7, T-47D, and colon tumor cell line HCT-8). Insulin receptors on breast tumor cells were resistant to down regulation (15-17% maximum loss of insulin binding after 4 h exposure to 170 nM insulin). HCT-8 cells were sensitive to down regulation after 4 h exposure to 3.8 nM insulin, but the extent of down regulation then lessened at higher concentrations of insulin. This paradoxical behavior was associated with increasing affinity of insulin receptors for insulin following exposure to hormone. Insulin-stimulated [3H]leucine incorporation into protein was measured in parallel with studies of receptor regulation to assess the effect of preexposure of cells to insulin on cell metabolism. Maximum down regulation of receptors in all three types of tumor cell by prior exposure to insulin did not significantly alter the responsiveness of any of the cell lines to insulin. Thus insulin receptor down regulation is abnormal in these tumor lines compared with reported studies in normal cells, and this may contribute a metabolic advantage to these malignant cells over normal tissues.

Breast Neoplasms↗

PubMed1987

Effects of metformin and glibenclamide on insulin receptors in fibroblasts and tumor cells in vitro.

The effects of the oral hypoglycemic agents metformin and glibenclamide on receptor binding of insulin and insulin-induced receptor down-regulation were studied in cultured normal human fibroblasts, human breast tumors (cell lines MCF-7 and T-47D) and a human colon tumor (cell line HCT-8) in order to identify differences in receptor regulation between normal and transformed cells. Binding of 125I-insulin was not significantly altered in any cell type following 24-h exposure of cells to concentrations of these agents equivalent to the in vivo therapeutic range, either in the presence or absence of insulin. Glibenclamide (2 microM) and metformin (1-10 microM) induced a 13-28% reduction in insulin receptor down regulation in fibroblasts exposed to 1.7 x 10(-8)M-insulin, the loss of binding on exposure to insulin decreasing from 55% to 40-48%. Both agents induced a smaller percentage reduction in insulin-induced receptor down-regulation in HCT-8 cells exposed to 1.7 x 10(-8)M insulin for 4 h (26% binding loss reducing to 14-20% in the presence of drug) and in MCF-7 and T-47D cells exposed to 1.7 x 10(-7)M insulin for 4h (16% binding loss reducing to 10%). It remains to be shown whether these small differences in sensitivity of receptors in malignant and normal cells to regulation by insulin and oral hypoglycemic agents can be exploited clinically in cancer management programs.

Breast Neoplasms↗

PubMed1987

Cytokinetic factors in drug resistance of Lewis lung carcinoma: comparison of cells freshly isolated from tumours with cells from exponential and plateau-phase cultures.

The cytotoxic effects of chemotherapeutic drugs on quiescent and actively proliferating cells of a Lewis lung carcinoma (LLTC) cell line have been examined. The sensitivities of cells in plateau-phase and exponentially growing cultures were compared with those of cells recovered from large subcutaneous tumours both immediately after tumour disaggregation and after one or 4 days in culture. Flow cytometric analysis indicated that when cells freshly prepared from tumours were placed into culture, they underwent extensive recruitment into S-phase. Several drugs were less cytotoxic towards both plateau-phase cultured cells and cells freshly isolated from tumours than they were against exponentially growing cells. These included amsacrine, its 4-methyl-5-(N-methyl)carboxamide derivative CI-921, doxorubicin, and nitrogen mustard. In contrast to these drugs, chlorambucil and plasma from cyclophosphamide-treated mice did not show decreased activity against slowly proliferating cells from cultures or tumours relative to cells in an actively proliferating state. The similar sensitivities of plateau-phase cultured cells and cells taken directly from large growing tumours is direct evidence that plateau-phase cultures are a useful approximation to the state of cytokinetic resistance to chemotherapeutic drugs that prevails in solid tumours, although they may not fully reflect the cytokinetic heterogeneity present in tumours.

Amsacrine↗

PubMed1987

Resistance of cultured Lewis lung carcinoma cell lines to tiazofurin.

An established in vitro cell line (LLTC), originally derived from the Lewis lung carcinoma (LL), was found to have lost sensitivity to the C-nucleoside antitumor agent tiazofurin (NSC-286193; 2-beta-D-ribofuranosylthiazole-4-carboxamide) both in vitro and in vivo. A new in vitro cell line (LLAK) was derived from LL and compared to LLTC in its growth properties and sensitivity to tiazofurin. LLAK resembled the in vivo tumor in having both a high S-phase fraction and a high rate of cell death at high cell density. In continuous drug exposure growth inhibition assays, the concentration of tiazofurin required to reduce the number of cells in a culture by 50% with respect to control cultures was 0.51 microM for LLAK, 2.6 microM for LLTC, and greater than 10 microM for a range of human cancer cell lines. In cytotoxicity assays involving a 2-hour drug exposure followed by clonogenic assay, tiazofurin was more toxic to LLAK cells than to LLTC cells or L1210 murine leukemia cells, consistent with its high in vivo activity against LL. MM-96 human melanoma cells were highly resistant. Flow cytometry studies indicated that tiazofurin selectively depleted the LLAK cell population of S- and G2-phase cells. In one experiment involving 16 consecutive in vitro passages of LLAK in the absence of tiazofurin, a new line emerged that was resistant to tiazofurin in the clonogenic assay. The results demonstrate the spontaneous emergence of resistance from a tiazofurin-sensitive cell line. If similar processes occur during adaptation of human tumor cells to culture, this may explain the finding of low activity of tiazofurin toward a range of human tumor cell lines.

Animals↗