PubMed Health⌕ Search

Biomedical subjects

K J Scanlon

Publications and source records attributed to K J Scanlon.

At least 55 records · Page 3Linked to original sources
PubMed1992

The role of folates in the development of methotrexate resistance in human leukemia cell line K562.

The effect of reduced and oxidized folates on the development of methotrexate (MTX) resistance has been examined in human leukemia cell line K562 (K562/S). K562/S cells were made resistant to MTX by soft-agar cloning either in RPMI-1640 medium (K562/MTX-PGA) or in folic-acid-free RPMI-1640 medium containing 10 nM leucovorin (K562/MTX-LV). The optimal concentrations of leucovorin for the growth of K562/S, K562/MTX-PGA and K562/MTX-LV cells were 1 nM, 5 nM and 10 nM respectively. K562/MTX-PGA cells were 24-fold resistant to MTX as noted by impaired MTX transport. In contrast, K562/MTX-LV cells were 26-fold resistant to MTX as noted by gene amplification of dihydrofolate reductase. Furthermore cross-resistance to cytosine arabinoside was only demonstrated in K562/MTX-PGA, while the K562/MTX-LV cells showed no significant cross-resistance to cytosine arabinoside. These results suggest that the type and level of folates used during the development of MTX resistance may play a role in the mechanism for MTX resistance. Leukemia cells that are grown in leucovorin might serve as a model for acquired MTX resistance in vivo.

Cell Cycle↗
PubMed1992

The utility of an anti-fos ribozyme in reversing cisplatin resistance in human carcinomas.

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMTII-A, genes previously linked to DNA synthesis and repair. Thus these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP syntase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.

Antineoplastic Agents↗
PubMed1991

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.

Base Sequence↗
PubMed1991

Cisplatin resistance in human cancers.

Cancer chemotherapeutic agents primarily act by damaging cellular DNA directly or indirectly. Tumor cells, in contrast to normal cells, respond to cisplatin with transient gene expression to protect and/or repair their chromosomes. Repeated cisplatin treatments results in a stable resistant cell line with enhanced gene expression but lacking gene amplification for the proteins that will limit cisplatin cytotoxicity. Recently, several new human cell lines have been characterized for cisplatin resistance. These cell lines have led to a better understanding of the molecular and biochemical basis of cisplatin resistance. The c-fos proto-oncogene, a master switch for turning on other genes in response to a wide range of stimuli, has been shown to play an important role in cisplatin resistance both in vitro and in patients. Based on these studies, new strategies have been developed to circumvent and/or exploit clinical cisplatin resistance.

Animals↗
PubMed1990

Cyclosporin A suppresses cisplatin-induced c-fos gene expression in ovarian carcinoma cells.

Cisplatin-resistant A2780DDP human ovarian carcinoma cells, 1 and 3 h following cisplatin treatment, display 3.2-6.1-fold enhancement of mRNA expression for oncogenes c-fos and c-H-ras and for enzymes necessary for DNA synthesis and repair. In contrast, c-myc mRNA was partially decreased. Increased transcription for the aforementioned genes accounts for the higher levels of mRNA since the enhancement is susceptible to actinomycin D pretreatment. Cyclosporin A (CSA) has been shown to reverse resistance to a variety of antineoplastic agents. An 18-h treatment with CSA yielded a 6-fold improved sensitivity to cisplatin in A2780DDP cells. When A2780DDP cells are pretreated with CSA followed by cisplatin, there is no increase in mRNA for c-fos and c-H-ras and the genes for thymidine synthesis and DNA repair. Their expression approaches the levels demonstrated in A2780S cells. In contrast, c-myc mRNA was elevated 7.4-fold in the presence of CSA in A2780DDP cells. Long term weekly exposures of A2780DDP cells to CSA resulted in the evolution of a revertant cell line, A2780DDP/CSA, with decreased c-fos and dTMP synthase mRNA. Thus, cisplatin treatment in A2780DDP cells differentially induces expression of certain nuclear oncogenes (c-fos/c-myc), as well as genes possibly involved in the repair of cisplatin-mediated DNA damage, an induction suppressed by CSA treatment.

Cisplatin↗
PubMed1990

Heat radiosensitization and the level of DNA polymerases alpha and beta of human colony-forming unit-granulocyte-macrophage and myeloid leukemias sensitive and resistant to chemotherapeutic agents.

In these studies, heat radiosensitization in normal human colony-forming unit-granulocyte-macrophage (CFU-GM) and several different leukemic cell lines sensitive or resistant to chemotherapeutic agents were measured. Extent of heat radiosensitization was then correlated with the level of DNA polymerases alpha and beta in control and heat-shocked cells in order to examine whether there is a positive correlation between the degree of heat radiosensitization and the level of these enzymes. Our results show that human bone marrow CFU-GM have an x-ray response with D0 of 1.56 Gy and a small amount of heat radiosensitization with a thermal enhancement ratio (TER) of 1.2. K562, a human erythroleukemic cell, showed a D0 of 1.32 +/- 0.2 Gy and TER of 1.4. However, in contrast to normal CFU-GM which showed no shoulder in the X-ray survival curve, K562 cells showed a small shoulder with a quasi-threshold dose, (Dq) of 2 Gy and n of 2. K562 cells resistant to chemotherapeutic drugs such as 1-beta-D-arabinofuranosylcytosine and etoposide (VP-16) showed D0 of 1.47 +/- 0.13, and 1.77 +/- 0.18 Gy; Dq of 4 and 0 Gy; and n of 5 and 1; and TER of 1.6 and 2, respectively. The level of DNA polymerases alpha and beta activity and their respective mRNA levels were approximately the same in all cells. The reduction in the level of DNA polymerase beta after heat treatment however, correlated with the TER obtained for various leukemic cells. These studies indicate that normal CFU-GM and variety of human leukemic cells show only a small amount of heat radiosensitization. However, drug-resistant leukemic cells show a higher amount of heat radiosensitization than their drug-sensitive parent line. This suggests that hyperthermia may be beneficial in eradicating drug-resistant leukemic cells when combined with X-ray. Furthermore, the inactivation of DNA polymerase beta activity results in a higher amount of heat radiosensitization.

Blotting, Northern↗
PubMed1990

Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil.

Peritoneal cells were derived from a patient (PK) with adenocarcinoma of the colon during the course of cisplatin/5-fluorouracil (5-FUra) treatment. Resistance to cisplatin and 5-FUra, characterized by a lack of response to chemotherapy and continued growth of the tumor, was concomitantly associated with a 2-4-fold increase in DNA copy number for dTMP synthase and dihydrofolate reductase. There was a corresponding amplification in DNA copy number of the c-myc (2X), H-ras (4X), and c-fos (15X) oncogenes. Cytogenetic studies revealed an iso (13q) chromosome, but failed to show any double minutes or homogeneously staining regions. In addition, drug-resistant tumor cells from PK and another patient (HG) displayed enhanced expression of dTMP synthase, c-fos and DNA polymerase beta when compared to normal colon tissue and the HCT8 human colon carcinoma cell line. These results suggest that elevated oncogene DNA and gene expression may be involved in the development of cisplatin resistance.

Adenocarcinoma↗
PubMed1990

Cyclosporin A suppresses cisplatin-induced oncogene expression in human cancer cells.

Most cancer chemotherapeutic agents are designed to damage DNA directly or indirectly. One mechanism of cellular resistance to these agents is enhanced DNA repair. Human ovarian carcinoma cells resistant to cisplatin (A2780DDP) respond to cisplatin treatment with a 3-6-fold increase in gene expression for oncogenes, DNA repair enzymes and enzymes necessary for the synthesis of thymidine. Cyclosporin A has been shown to reverse drug resistance, but its mechanism of action is unclear. In this study, weekly exposures of A2780DDP cells to cyclosporin A resulted in the evolution of a revertant cell line A2780DDP/CSA that was sensitive to cisplatin again and suppressed the induction of genes necessary for the repair of drug-induced DNA damage.

Cisplatin↗
PubMed1990

Potentiation of azidothymidine cytotoxicity in cisplatin-resistant human ovarian carcinoma cells.

The combination of cisplatin and AZT was synergistic in a subline (A2780DDP) of human ovarian carcinoma cells resistant to cisplatin, in contrast to the parental carcinoma cell line, A2780S. A2780DDP cells have elevated levels of enzymes necessary for DNA synthesis and repair. If A2780DDP cells respond to cisplatin with an increase in thymidine kinase activity, then AZT may chain terminate newly synthesized DNA. To test this hypothesis, a dual label [( 14C]-thymidine/[3H]-AZT) experiment was designed. A2780 cells were first incubated with [14C]-thymidine to label DNA and measure DNA degradation in response to cisplatin. These [14C]-thymidine labeled A2780 cells were then incubated for short intervals with [3H]-AZT to measure chain termination after cisplatin addition. A2780S cells responded to cisplatin with a modest increase in thymidine turnover and an increase in AZT incorporation. In contrast, A2780DDP cells initially responded to cisplatin treatment with a significant increase in thymidine turnover and a corresponding increase in [3H]-AZT incorporation. This was concomitantly associated with an increase in thymidine kinase mRNA within 2 hr after cisplatin treatment. Thus, the A2780DDP cells had the ability to rapidly turnover DNA, a property effectively exploited by the deoxythymidine analogue, AZT, utilizing the enhanced enzymes of the DNA synthesis and repair complex in A2780DDP cells.

Cell Line↗
PubMed1989

Utility of the polymerase chain reaction in detection of gene expression in drug-resistant human tumors.

Recently, an enzymatic amplification method, the polymerase chain reaction (PCR), was modified to amplify a sequence of a drug resistance gene. The PCR assay can confirm data achieved by conventional molecular biology techniques, while requiring less time and fewer patient cells. It can be quantitated for gene expression. The data generated make it possible to analyze m-RNA expression in tumor samples without being limited to detecting only gene amplification in response to cancer chemotherapy. The PCR assay can be an effective device in the early detection of resistance to chemotherapy.

Antineoplastic Agents↗
PubMed1989

Drug selectivity and membrane transport properties of normal human lymphocytes.

This study used Interleukin (IL)-stimulated normal human lymphocytes in culture as a model system to examine membrane transport systems and drug cytotoxicity. Normal peripheral blood mononuclear cells were stimulated with either purified IL-1 or phytohemagglutinin (PHA) and maintained for several months in IL-2. Only 'T' lymphocytes grew in culture. Amino acid transport was compared in PHA-stimulated cells and IL-stimulated cells. Both the PHA- and IL-stimulated lymphocyte cell lines had similar generation times and similar uptake and efflux of cisplatin and methotrexate. Yet, PHA-stimulated cells proved 3- to 5-fold more sensitive to the cytotoxic effects of these drugs. The greater drug sensitivity of the PHA-stimulated cell line was associated with enhanced sodium-dependent methionine transport and altered amino acid transport systems. Differences in the nutrient transport systems are comparable to those previously observed between drug sensitive and resistant cells, and may provide the biochemical basis for cisplatin and methotrexate collateral resistance.

Amino Acids↗
PubMed1989

Overexpression of DNA replication and repair enzymes in cisplatin-resistant human colon carcinoma HCT8 cells and circumvention by azidothymidine.

Biochemical differences were demonstrated between two cell lines derived from a human colon carcinoma (HCT8), one sensitive (HCT8S), and one 4.3-fold resistant to cisplatin (HCT8DDP). The cisplatin-resistant cell line overexpressed five enzymes (dihydrofolate reductase, thymidine 5'-monophosphate synthase, thymidine kinase, and DNA polymerase alpha and beta) believed to be important for DNA replicative and repair synthesis. In addition, the c-fos and c-H-ras oncogenes were also overexpressed in the HCT8DDP cells. This apparent overexpression was not associated with increases in gene copy number, it was related, however, to increased mRNA content. Expression of these key enzymes may be a significant factor in the development of clinical resistance to cisplatin. Further, these specific changes in cellular metabolism associated with cisplatin resistance may be exploited by the use of nucleoside analogues.

Cell Line↗
PubMed1988

Detection of drug resistance in human tumors by in vitro enzymatic amplification.

Both acquired and natural resistance to chemotherapy agents have proved problematic in the treatment of neoplasia. Thymidylate synthase, which catalyzes the synthesis of thymidine precursors, has been shown to be amplified in response to a variety of chemotherapeutic agents. The detection of such amplification could prove beneficial in the development of alternative clinical protocols. In this study we report the use of existing enzymatic amplification methods in order to detect incipient amplification of the thymidylate synthase gene upon resistance to cisplatin. The assay utilizes a modification of the polymerase chain reaction in which a sequence of the thymidylate synthase gene is amplified including two flanking oligonucleotides acting as primers for DNA synthesis. This method exhibits greater sensitivity than conventional nucleic acid detection methods and requires less than 100 ng of total RNA from patient tumors and no in vitro culturing of patient cells.

Cell Line↗
PubMed1988

Biochemical and molecular properties of cisplatin-resistant A2780 cells grown in folinic acid.

In this study, A2780 human ovarian carcinoma cells were grown in folinic acid in contrast to folic acid, and the molecular and biochemical properties of cisplatin-resistant A2780 cells were analyzed for changes in the dTMP synthase cycle. At concentrations of folinic acid that were optimal for cell growth (10(-8) M), the ED50 for cisplatin was 2.5 and 43 microM in the A2780S and A2780DDP cells, respectively. Resistance to cisplatin was associated with a 2-fold cross-resistance to 5-fluorodeoxyuridine and 5-fluorouracil as well as a 3-fold increase in both dTMP synthase activity and mRNA. The ED50 for methotrexate was similar in both A2780S and A2780DDP cells (1.2 microM). When both the A2780S and A2780DDP cells were grown in folinic acid, there was no significant difference in the level of dihydrofolate reductase activity. This data would suggest that cisplatin resistance is associated with changes in folate metabolism.

Cell Line↗
PubMed1988

Mechanisms of cross-resistance to methotrexate and 5-fluorouracil in an A2780 human ovarian carcinoma cell subline resistant to cisplatin.

Some properties of the human ovarian carcinoma line A2780 and a subline three times more resistant than the parent line to cisplatin are compared in this report. The rates of uptake and release of cisplatin were similar in the two cell lines. Resistance to cisplatin was associated with: (a) cross-resistance to 5-fluorouracil and methotrexate; (b) a 2.5-fold increase in thymidylate synthase, as measured by both enzyme activity and the capacity to complex 5-fluorodeoxyuridylate; and (c) an increase in the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate. These data suggest that cross-resistance to 5-fluorouracil and methotrexate in A2780 cells may be a consequence of increases in their respective target enzymes.

Biological Transport↗
PubMed1988

Mechanisms for cisplatin-FUra synergism and cisplatin resistance in human ovarian carcinoma cells both in vitro and in vivo.

Cisplatin and FUra act synergisticly in human carcinomas. An increase in the availability of reduced folates necessary for tight binding of FdUMP to thymidylate synthase (TS) contributes to the enhanced cytotoxicity of this drug combination. The human ovarian A2780 cell line made three-fold resistant to cisplatin has been shown to have a three-fold elevation of m-RNA for dihydrofolate reductase (DHFR) and TS. However, this increase did not result from an amplification of the genes for these two enzymes. In contrast, ovarian carcinoma cells from patients who failed treatment with cisplatin and FUra have been shown to have both enhanced gene expression and increased gene copy number for DHFR and TS.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1988

Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells.

Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+-oxidoreductase, EC 1.5.1.3) and thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence of enhanced expression of the thymidylate synthase cycle.

Animals↗