Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.     

Schedule-dependent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines

We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox-->Pacl) induces a synergistic cytotoxic effect in the BRC-230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox-->Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox-->Pacl sequence in a preclinical study. Our study was performed on BRC-230 and MCF-7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinko's test. When Gem (0.01 microg/ml for 24 hr) was given immediately or 24 hr after Dox-->Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox-->Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G2-M (95% in treated vs. 21% in control samples) 24 hr after Dox-->Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G1-S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule- and time-dependent and should be taken into consideration in the planning of clinical protocols.     

Sensitization to doxorubicin resistance in breast cancer cell lines by tamoxifen and megestrol acetate

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.     

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.     

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.     

Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines

A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium.     

Mechanisms of resistance to therapy and tumor cell survival

The failure to cure patients with cancer continues to be primarily because of the development of treatment resistance. Both normal and malignant cells die by either programmed cell death (apoptosis) or by cytolysis. Malignant cells have developed mechanisms of resistance that prevent them from entering the programmed cell death pathway as well as mechanisms of escaping immune recognition and cytolysis. These mechanisms include specific protective adaptations involving drug transport, metabolism, and target interactions. Malignant cells may also become resistant to therapy through alterations in genes encoding proteins involved in the initiation of apoptotic pathways. Finally, tumor cells may develop mechanisms to escape immune recognition, making them resistant to T-cell destruction. This article provides an overview of these mechanisms, with emphasis on published articles reported within the past year.     

Relationship between amine-carboxyboranes and TNF alpha for the regulation of cell growth in different tumor cell lines

The amine-carboxyboranes were shown to be synergistic with tumor necrosis factor alpha (TNF alpha) in cytotoxicity and inhibition of DNA synthesis in select types of cancer cells depending on the presence of a TNF alpha high affinity receptor on the membrane of the cell. Initially both TNF alpha and the amine-carboxyboranes reduce the influx of calcium but later cause a significant increase intracellularly. This influx is not linked with the amine-carboxyborane activating the calcitonin receptor in the tumor cells. Neither the agents nor TNF alpha directly inhibits DNA topoisomerase II activity but both did cause decreased phosphorylation of the enzyme by protein kinase C (PKC). The two agents caused synergistic inhibition. This event correlated with increased DNA protein linked breaks, DNA fragmentation and cell death. These protein linked breaks are additive with etoposide's effects but the latter agent's mechanism is different than phosphorylation of topoisomerase II. There was no evidence that the DNA fragmentation was caused by a calcium induced endonuclease enzyme in these cancer cells. The low-molecular weight amine-carboxyboranes appear to play an identical function as TNF alpha in its role to cause DNA breaks and fragmentation to cause apoptosis.     

Differentially potentiating effects by dipyridamole on cytotoxicity of 5-fluorouracil against three human maxillary cancer cell lines derived from a single tumor

Twenty specimens of six species of Nile fishes were examined for the presence of fungi. Of which 2 were from Alestes nurse; 3 from Bagrus docmac; 4 from Barbus bynni; 6 from Chrysichthys auratus; 4 from Lates niloticus and 1 from Malapterurus electricus. Forty-three fungal species in addition to 1 variety appertaining to fifteen genera were recovered from skin (15 genera and 34 species + 1 variety); gills, kidney (12 genera and 30 species + 1 variety, each); liver (11 genera and 30 species + 1 variety) and intestine (13 genera and 30 species + 1 variety) of all specimens, using glucose Czapek-Dox medium at 28 degrees C. The most common genera were Aspergillus, Penicillium and Trichoderma.     

Immunologic characterization of tumor markers in human ovarian cancer cell lines

OBJECTIVE: The purpose of this study was to evaluate the expression of a novel autologous ovarian tumor-associated antigen in eight human ovarian tumor cell lines compared with other ovarian tumor markers and products of oncogenes. METHODS: Autologous antibodies were eluted from human ovarian tumor-membrane fragments purified in our laboratory. These antibodies react with autologous ovarian tumor-associated antigens (AOTA) and have a high degree of specificity for human ovarian tumors. They do not bind to normal ovarian or nonovarian tissues, or to nonovarian neoplastic tissues. We evaluated eight human ovarian adenocarcinoma cell lines (2008, 2774, Caov-3, OVCAR-3, PA-1, SW 626, UCI 101, and UCI 107) by indirect immunofluorescence to determine the expression of AOTA relative to the ovarian cancer tumor marker CA 125 and the products of selected oncogenes (p 53, c-neu, and c-myc). RESULTS: The patterns and intensities of immunofluorescence correlated most closely between AOTA and c-neu. For example, AOTA and c-neu were detected in all eight cell lines and displayed very strong cytoplasmic fluorescence on cell lines 2774, UCI 101, and UCI 107. CA 125 was present in the cytoplasm of four of eight cell lines. A tumor suppressor gene product, p53, exhibited a nuclear staining pattern in six of eight cell lines. CONCLUSIONS: These data suggest that AOTA and the products of the c-neu oncogene may share certain epitopes. Current studies are underway to increase our understanding of the humoral response to ovarian cancer and the possible relationship to the expression of tumor oncogene products. Further characterization of AOTA will be necessary before early diagnostic tests can be developed.     

Increased activity and sensitivity of mitochondrial respiratory enzymes to tumor necrosis factor alpha-mediated inhibition is associated with increased cytotoxicity in drug-resistant leukemic cell lines

The drug-resistant leukemic cell lines, CEM/VLB100 and K/DAU600, are more sensitive to tumor necrosis factor alpha (TNFalpha)-mediated cytotoxicity compared with their parental cell lines, CCRF-CEM and K562 cl.6. Drug-resistant leukemic cell lines have more active mitochondrial function, which is associated with a greater susceptibility to TNFalpha-induced respiratory inhibition. TNFalpha blocked electron transfer at three sites, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Respiratory rate and electron transport chain enzyme activities were significantly inhibited in the drug-resistant, TNF-sensitive cell lines. Respiratory inhibition preceded cell death by at least 5 to 8 hours. The respiratory failure was not compensated for by appropriate up-regulation of the glycolytic pathway. Increasing mitochondrial respiratory rate and enzyme activities by long-term culture with 2 mmol/L adenosine 5'-diphosphate (ADP) and Pi sensitized both drug-sensitive and drug-resistant cells to TNFalpha-induced cytolysis. Intramitochondrial free radicals generated by paraquat only had a limited and delayed effect on respiratory inhibition and cytolysis in comparison with the effect of TNFalpha. We conclude that TNFalpha-induced cytotoxicity in leukemic cells is, at least in part, mediated by inhibition of mitochondrial respiration. Free radical generation by TNFalpha may not directly lead to the observed inhibition of the mitochondrial electron transport and other mechanisms must be involved.     

Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer

Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex hormones in lung cancer, the expression of sex-steroid receptors and the glucocorticoid receptor was investigated in 29 lung-cancer cell lines stemming from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) by means of immunocytochemistry, ligand-binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of investigation, NSCLC cell lines showed a low expression of estrogen receptor in 6, progesterone receptor in 13 and androgen receptor in 12 out of 17 cases examined; sex-steroid-receptor expression was virtually absent in SCLC cell lines. The glucocorticoid receptor was expressed in all 29 cell lines studied. Additionally, 52 tumor samples from primary lung cancer were investigated for their receptor expression by means of immunohistochemistry. Among patients with primary lung-cancer sex-steroid-receptor expression in tumor biopsies was detected most frequently in female patients (in 69% of 16 cases, vs. 42% of 36 tumors from men) and in patients with adenocarcinoma. Further research will focus on these subgroups. Immunohistology is a feasible method of studying steroid-receptor expression in lung cancer.     

Cell cycle dependency of 67gallium uptake and cytotoxicity in human cell lines of hematological malignancies

67Gallium (67Ga) is a radionuclide which accumulates in hematological malignancies and is used for diagnostic imaging. We investigated in this in vitro study the cell cycle dependency of cellular uptake and cytotoxicity of 67Ga. Cell cycle synchronization of cells was achieved by counterflow centrifugal elutriation and the use of cytostatic drugs. The human lymphoma cell lines U-937 and U-715 were used and in elutriation experiments we also used the leukemic cell line HL-60. The transferrin receptor (CD71) expression, 67Ga uptake and cell proliferation inhibition were the parameters measured. We also studied cytotoxicity in various schedules for combination of 67Ga and drugs and the residual proliferative capacity was measured. The CD71 expression in the three cell lines increased from 106-177% on S phase cells and from 118-233% on G2M cells, as compared to the G0/G1 cell fraction. The 67Ga uptake varied from 108-127% for S cells and 128-139% for G2M cells. The drugs chosen induced cell cycle phase accumulation in S and/or G2M phase during preincubation. 67Ga preincubation induced accumulation in the G2M phase. Almost all combinations of 67Ga and drugs resulted in a non-interactive effect, except for methotrexate which resulted in an antagonistic effect. No preferential effect of any of the incubation schemes was seen. CD71 expression and 67Ga uptake were increased in S and G2M cells. Combination of 67Ga with drugs which arrest cells in these cell cycle phases did not result in a change in cytotoxicity. However, these results implicate that 67Ga and the cytostatic drugs tested except for methotrexate might be used together or sequentially in therapy.     

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.     

Lymphocyte response to transitional cell carcinoma: peripheral cytotoxicity and local tumor infiltration

The cytotoxicity of peripheral lymphocytes from patients with transitional cell carcinoma was evaluated and the results were compared to the degree of local lymphocytic infiltration in the bed of the primary tumor. Although a clear correlation was not established local lymphocytic infiltration was associated more commonly with tumor-related peripheral lymphocyte cytotoxicity.     

Retroviral transfer of deoxycytidine kinase into tumor cell lines enhances nucleoside toxicity

Deoxycytidine kinase (dCK) phosphorylates a number of nucleoside analogues that are useful in the treatment of various malignancies. Although the level of dCK activity in malignant cells is thought to correlate with chemotherapeutic response, no direct data are available to support this assumption. We have tested this hypothesis by infecting three tumor cell lines, MCF-7, HT-29, and H1437, with the retroviral vector LNPO containing either dCK or LacZ cDNA and measuring the corresponding sensitivity to nucleoside analogues. DCK activity was increased by 1.7-, 2.3-, and 16-fold in MCF-7, HT-29, and H1437 cells, respectively. Northern and Western blots demonstrated a similar increase in mRNA and protein levels. As a result of dCK expression, MCF-7 cells demonstrated a 2.5-fold increase in drug sensitivity to 1-beta-D-arabinofuranosylcytosine (AraC) and 2-chloro-2'-deoxyadenosine (CdA). HT-29 cells had a 7-fold increase in sensitivity to AraC, CdA, and 2-fluoro-9-beta-D-arabinofuranosyladenine, whereas H1437 cells demonstrated a 20- to 106-fold increase. For all three drugs, there was a linear relationship between dCK activity in clonally derived cell lines and IC50s. These data demonstrate a direct effect of dCK activity on drug sensitivity in cell lines. Because many tumors have relatively low levels of dCK, it is possible that dCK gene transfer will be a useful adjunct to the treatment of these malignancies.     

Growth inhibitory effects of aromatic fatty acids on ovarian tumor cell lines

Epithelial ovarian cancer is a major cause of cancer-related mortality in women, making the search for new treatment modalities essential. Sodium phenylacetate (NaPa), a phenylalanine derivative, has been shown to induce cytostasis and differentiation by inhibiting protein isoprenylation. Similar effects have been observed with phenylbutyrate, a phenylacetate congener. We examined in parallel the growth inhibitory activity against human ovarian carcinoma cell lines of phenylacetate, phenylbutyric acid (PB), and certain related compounds, and comparisons were made with lovastatin. On a molar basis, hydroxykynurenine and kynurenine showed the highest activity followed by PB and NaPa. Ovarian carcinoma cell lines were also sensitive to lovastatin in micromolar concentrations. Additive effects were observed when PB was combined with cisplatin or when NaPa or PB were combined with lovastatin. NaPa and PB, but not kynurenine, inhibited incorporation of [3H]mevalonate into ovarian carcinoma cells. An immune modulatory role might also be suggested for PB because it resulted in increased ovarian tumor cell expression of human leukocyte antigen class I and the cluster of differentiation molecule CD58, whereas transforming growth factor-beta2 expression was decreased. Phenylbutyrate, which is the ester form of PB, has shown acceptable pharmacological properties and clinical responses in patients with other malignancies, and might be considered for evaluation in ovarian cancer.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Azidothymidine in combination with 5-fluorouracil in human colorectal cell lines: in vitro synergistic cytotoxicity and DNA-induced strand-breaks

The in vitro cytotoxicity of the combination of azidothymidine (AZT) and 5-fluorouracil (5-FU) against the human colorectal cancer cells SW-480, SW-620 and COLO-320DM was evaluated. The cytotoxic effects of 5-FU and AZT were determined by the assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenil)-2H-tetrazolium-5-carbo xanilide inner salt (XXT), while drug-induced DNA strand-breaks were measured using a fluorometric analysis of DNA unwinding. After an exposure of 72 h, 5-FU and AZT induced a dose-dependent cytotoxicity against each cell line. The addition of 3, 10 and 30 microM AZT to various concentrations of 5-FU, as well as the addition of 0.5, 1 and 3 microns 5-FU to various concentrations of AZT, resulted in an enhanced cytotoxic effect. Isobologram analysis and the combination index (CI) method demonstrated that the interaction between 5-FU and AZT was clearly synergistic in each cell line, except for the 30% level of effect in SW-620, where borderline synergism was observed. The evaluation of DNA strand-breaks after an exposure of 16 h to 5-FU, AZT or 5-FU + AZT demonstrated that the 5-FU + AZT combination produced the greatest DNA damage, and that this interaction was synergistic in each cell line. In conclusion, our study supports the evidence that the potential antitumour activity of AZT can be modulated by combining it with agents which inhibit thymidylate (dTMP) formation, such as 5-FU, and that the increased cytotoxicity is related to enhanced DNA damage. These findings should encourage further experimental and clinical studies of the potential use of AZT in combination with inhibitors of de novo dTMP synthesis.     

Cytotoxicity induced by Erythrina variegata serine proteinase inhibitors in tumor hematopoietic stem cell lines

A prospective study of activated protein C sensitivity, protein C, protein S, and other coagulation factors in 239 women during normal pregnancy was carried out. Protein C activity appeared unaffected by gestation, although an elevation of protein C activity was observed in the early puerperium. A fall in total and free protein S with increasing gestation was observed. Activated protein C sensitivity ratio (APC:SR) showed a progressive fall through pregnancy. This fall correlated with changes in factor VIIIc, factor Vc and protein S. 38% of subjects, with no evidence of Factor V Leiden or anticardiolipin antibodies, showed a low APC:SR (APC:SR <2.6) in the third trimester of pregnancy. Aside from a significant reduction in birth weight, no difference in pregnancy outcome was observed between these subjects and those with a normal APC:SR. Activated protein C sensitivity ratio, modified by pre-dilution of patient samples with factor V depleted plasma, showed no consistent trend with gestation.