Topoisomerase IIalpha and other drug resistance markers in advanced non-small cell lung cancer.

Resistance to chemotherapy is common in non-small cell lung cancer. The aim of this study was to investigate the prognostic impact of in vitro established drug resistance markers on the response to chemotherapy in patients with advanced non-small cell lung cancer. Samples of 38 patients were analyzed by immunohistochemical staining, for topoisomerase IIalpha and IIbeta, Ki-67, MRP and LRP. In addition, mutation analysis of the topoisomerase IIalpha gene, the B/DNBS and the Tyr804 region, was performed. Lung tumor biopsies were taken prior for treatment with one of the following regimens; cisplatin/paclitaxel, cisplatin/VM26 or VP16, or carboplatin/VP16/ifosfamide. Seventeen patients obtained a partial response, 12 had stable disease and nine patients had progressive disease. None of the investigated markers was related with overall response rate. In one sample a point mutation in the B/DNBS region of the topo IIalpha gene was detected which substitutes IIe(510) with Val. This tumor had a partial response to four courses of cisplatin/VP16 treatment. The survival analysis showed that the patients with high topo IIalpha expressing tumors had a significantly worse survival compared with the patients with low or intermediate topo IIalpha expressing tumors. In conclusion, no relation was observed between expression of topoisomerase IIalpha, IIbeta, Ki-67, MRP or LRP and response rate. Furthermore, worse survival was seen in patients with high topoisomerase IIalpha expressing tumors. In one tumor sample, a newly described mutation in the B/DNBS region of the topo IIalpha gene was detected, which does not appear to be related to drug resistance.

Validation of a polymerase chain reaction aided transcript titration assay (PATTY) for topoisomerase II in lung cancer samples.

This report describes the validation of a polymerase chain reaction aided transcript titration assay (PATTY) for tumor samples. The results obtained with the PATTY were compared to those of RNase protection in a set of 7 human lung cancer cell lines and in 23 non-small cell lung cancer samples derived from resected patients. Whereas between PATTY and RNase protection assay a good correlation was observed in the cell lines (r = 0.74, p = 0.057), no correlation was observed within the tumor samples (r = 0.06, p = 0.78). This was also the case when only tumors with a high percentage of tumor cells (> 90%) were selected. Although PATTY is a valuable tool to measure mRNA expression in cell lines, our results caution the use of PATTY in human tumor samples without proper validation. The possible causes of these results are discussed.

Characterization of human soft-tissue sarcoma xenografts for use in secondary drug screening.

We have established ten transplantable human soft-tissue sarcoma (STS) xenografts grown as subcutaneous tumours in the nude mouse. Nine xenografts originated from patients that needed chemotherapy in the course of their disease. The xenografts were tested for their sensitivity to maximum tolerated doses of five anti-cancer agents. Growth of treated tumours was expressed as a percentage of control tumour growth and a growth inhibition > 75% was measured for doxorubicin in 20% of the STS xenografts, for cyclophosphamide in 30%, for ifosfamide in 20%, for vincristine in 20%, whereas etoposide was not effective in the STS xenografts. In three out of ten STS xenografts MDR1 mRNA was detectable, but this was not related to the resistance against doxorubicin, vincristine or etoposide. Topoisomerase IIalpha mRNA expression levels did not reflect sensitivity to doxorubicin or etoposide. In all STS tissues, however, these levels were lower than topoisomerase IIalpha mRNA in a drug-sensitive human ovarian cancer xenograft. Glutathione concentrations and the activities of glutathione S-transferase, glutathione peroxidase and glutathione reductase were not related to resistance against the alkylating agents or doxorubicin. Of interest, in all STS tissues, glutathione S-transferase pi was the predominant isoenzyme present. In conclusion, chemosensitivity of the STS xenografts reflects clinical response rates in phase II trials on the same compounds in adult STS patients. Relatively low levels of topoisomerase IIalpha mRNA may partly account for intrinsic resistance against, for example, doxorubicin. Additional factors must contribute to moderate responsiveness to alkylating agents.

Determinants of CPT-11 and SN-38 activities in human lung cancer cells.

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.

Effects of tubulin-inhibiting agents in human lung and breast cancer cell lines with different multidrug resistance phenotypes.

Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP). All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines. In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells. Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times. In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil. MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect. These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells. However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.

CPT-11 sensitivity in relation to the expression of P170-glycoprotein and multidrug resistance-associated protein.

The relevance of P170-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) for the sensitivity to CPT-11 was investigated in human malignant cell lines as well as in human tumour xenografts. In vitro, the P-gp-positive sublines BRO/mdr1.1 (transfected with MDR1) and 2780AD were slightly cross-resistant against carboxylesterase-activated CPT-11. Cross-resistance against SN-38 was present in 2780AD cells, but not in BRO/mdr1.1 cells. The P-gp modulators BIBW22BS, verapamil and dexniguldipine partly reversed the resistance against CPT-11 in the P-gp-positive sublines. BIBW22BS was the most effective modulator in the reversal of the resistance against carboxylesterase-activated CPT-11 as well as against SN-38 in the 2780AD subline. In contrast to doxorubicin and vincristine, the BRO/mdr1.1 xenografts were at least as sensitive to CPT-11 as the BRO xenografts. The 2780AD xenografts were slightly less sensitive than the parent tumours, but there was no difference in topoisomerase I DNA unwinding activity. Therefore, the high retention of the multidrug-resistant phenotype of 2780AD cells in vivo may be the cause of the low cross-resistance against CPT-11. The MRP-positive subline GLC4/ADR was cross-resistant against carboxylesterase-activated CPT-11 and SN-38. GLC4/ADR cells, however, demonstrated a twofold lower topoisomerase I activity than GLC4 cells. Cross-resistance against the camptothecin derivatives was not apparent in the MRP-transfected subline of SW1573/S1. In conclusion, P-gp-positive cells show a low cross-resistance against CPT-11/SN38, which is only apparent with high P-gp expression in vivo. MRP does not seem to play a role in the sensitivity to CPT-11.

Anti-tumor activity of CPT-11 in experimental human ovarian cancer and human soft-tissue sarcoma.

CPT-11, a semi-synthetic derivative of camptothecin, was investigated for its activity in panels of 15 human ovarian-cancer lines and 10 human soft-tissue sarcoma lines grown s.c. in nude mice. Various factors were analyzed that may be of influence on the extent of tumor-growth inhibition induced by CPT-11. At equitoxic doses, CPT-11 was more effective in the daily x5 schedule than the weekly x2 schedule, although a 2-fold higher dose was administered in the weekly x2 schedule. Since i.p. and i.v. injections were similarly effective, the selected treatment schedule was 20 mg/kg i.p. daily x5, starting when tumors measured approximately 150 mm3. Growth inhibition of > or = 75% was obtained in 8/15 human ovarian-cancer lines and in 6/10 human soft-tissue sarcoma lines. A weak correlation was found between topoisomerase-I mRNA in xenograft tissues and sensitivity to CPT-11. Relative topoisomerase-I expression was highest in MRI-H-207 and WLS-160 xenografts, in which CPT-11 was able to induce cures of all tumors. The high efficacy in the 2 panels of human tumor lines suggests over-prediction of its potential clinical activity in these tumor types. The difference in efficacy of CPT-11 between species may be related to the metabolism of the drug, since CPT-11 is converted more efficiently into SN-38 in mice. In addition, mice may be less sensitive to SN-38-induced side-effects. On the basis of the preclinical data, frequent administration of lower doses of CPT-11 should be considered in order to increase response rates in the clinic.

Prognostic value of the expression of p53, bcl-2, and bax oncoproteins, and neovascularization in patients with radically resected non-small-cell lung cancer.

PURPOSE: To assess the prognostic value of p53, bcl-2, bax, and neovascularization in radically resected non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: Tumors from 116 patients were assessed by immunohistochemistry for expression of p53 (DO7 and PAb1081), bcl-2, and the quantification of microvessel density (CD-31). In addition, the expression of bax was assessed in 61 stage I tumors. The median levels of expression of each marker were used as cutoff points. RESULTS: p53 was not correlated to any patient or tumor characteristic, whereas bcl-2 showed higher expression in squamous cell carcinomas (P < .001). bax expression was significantly related with male sex (P = .006) and adenocarcinoma type (P = .0013). p53 status, assessed with one monoclonal antibody (MoAb), was not predictive for survival; however, the combination of staining results obtained with two MoAbs identified the DO7-/PAb1801+ tumors as those with the worst prognosis. bcl-2 expression was associated with longer survival in stage I patients (P = .0169). The combined group expressing p53+(PAb1801)/bcl-2- had the worst survival in stage I patients (P = .034) and in the whole series in comparison with the other combinations of the two oncoproteins. bax expression alone had no influence on survival of stage I patients, but patients with bax+/bcl-2- tumors had the worst prognosis (P = .02 in comparison with bax+/bcl-2+). Tumor neovascularization was not related with other factors, and patients with CD-31+ tumors had a shorter survival duration than those with CD-31- tumors only in stage II (P = .0283). By multivariate analysis including all patients, the presence of p53+/ bcl-2- tumor expression and large tumor diameter (> or = 4cm) were independent prognostic factors for shorter survival duration. For stage I, only the presence of bax+/ bcl-2- tumor expression had a significant negative influence on survival. CONCLUSION: The interaction and the regulation of new biologic markers, such as those involved in the apoptotic pathway, are complex. Combinations of the expression of several of them may give more valuable information than the study of just one. Prognostic influence of p53 staining varied depending on the choice of antibody and the combination of bcl-2- together with p53+ (PAb1801) or with bax+ had the worst influence on survival for patients with stage I NSCLC.

Expression of drug resistance proteins in breast cancer, in relation to chemotherapy.

Drug resistance plays an important role in chemotherapy failure in breast cancer. We studied the expression of MDR1, MRP, LRP, DNA topoisomerases, p53 and Ki-67 in different groups of breast cancer patients in relation to chemotherapy. Tissues from 6 normal breasts and 20 primary operable, 40 locally advanced and 10 anthracycline-resistant metastatic breast cancers were assessed. Sequential samples of the same patient were available from 17 patients with locally advanced breast cancer undergoing neo-adjuvant chemotherapy and in 7 metastatic patients undergoing paclitaxel treatment. Protein expression was investigated by immunohistochemistry. Significantly higher protein expression was observed for Pgp, Ki-67 and p53 in the locally advanced breast cancers than in primary operable breast cancers. No other significant differences in protein expression were found among the 3 breast cancer groups. Expression of none of the markers that could be assessed (Pgp, MRP, LRP, p53 and Ki-67) in locally advanced breast cancer had predictive value for pathological response. Interestingly, after chemotherapy a significant decrease in percentage of Ki-67 positive tumor cells was observed, whereas the other markers did not vary substantially. Furthermore, considering all breast cancer samples, a cumulative dose of doxorubicin >400 mg/m2 inversely correlated with Ki-67 positivity. However, 2 patients with a pathological complete remission had only 5-10% Ki67-positive tumor cells before chemotherapy, indicating that Ki67 negativity itself is not responsible for chemoresistance. In conclusion, none of the known proteins related to multidrug resistance predicted response to chemotherapy in breast cancer, and resistant clones left behind generally had a low proliferation rate.

Expression of the human major vault protein LRP in human lung cancer samples and normal lung tissues.

BACKGROUND: The recently discovered LRP protein has been shown to be involved in drug resistance and possibly in detoxification processes. MATERIALS AND METHODS: To study the relation between LRP expression and exposure to cigarette smoke, LRP immunoreactivity was evaluated in 39 paraffin embedded normal lung tissues derived from patients operated on for pneumothorax, and related to amount of pack years smoked. We also studied the LRP protein expression in 36 non-small-cell lung cancer (NSCLC) samples and related the expression to patient characteristics and survival. Furthermore 17 lung tumor samples (10 NSCLC and 7 SCLC) derived from patients treated with chemotherapy were analysed in order to investigate the relation between LRP or MRP expression and the patient's response to chemotherapy. RESULTS: In the normal lung tissues, LRP intensity levels were not correlated to the amount of pack years smoked, although a trend was seen for higher LRP intensity levels in patients who smoked more than 10 pack years. LRP expression was significantly higher in NSCLC samples than in SCLC samples, and all SCLC samples displayed very low LRP expression. Within NSCLC, squamous cell and adenocarcinomas had higher LRP expression than large cell undifferentiated and mixed tumors. In NSCLC patients LRP expression was not a prognostic factor for survival. At initial analysis LRP expression levels did not predict for the response to chemotherapy. Only 3 out of 17 patients expressed MRP, and all SCLC samples were MRP negative. CONCLUSIONS: Striking different expression levels were seen between NSCLC and SCLC for both LRP and MRP. In a preliminary analysis LRP expression was not predictive for response to chemotherapy in lung cancer patients. In pneumothorax patients LRP levels were not correlated with the amount of pack years smoked.

MRP is frequently expressed in human lung-cancer cell lines, in non-small-cell lung cancer and in normal lungs.

The multidrug resistance-associated protein (MRP), a new membrane transporter related to non-Pgp multidrug resistance, is overexpressed in some drug-selected cancer-cell lines. The role of MRP in unselected cell lines and in human cancer is unknown. MRP gene expression, determined by RNase protection assay and chemosensitivity to doxorubicin, etoposide and cisplatin, determined by MTT assay, were assessed in 18 non-drug-selected lung-cancer cell lines (10 small-cell lung cancer, 6 non-small-cell lung cancer, and 1 carcinoid). MRP gene expression was also investigated in normal lung tissue and primary non-small-cell lung cancer. All cell lines except one and all normal lung tissues and primary non-small-cell lung cancers expressed detectable levels of MRP. Expression was significantly lower in cell lines than in normal and neoplastic lung. MRP protein expression was also assessed by immunohistochemistry using the monoclonal antibody MRPr1; comparable levels of expression were observed between mRNA and protein in cell lines; however, in tumor samples intense staining was observed in tumor cells as well as in infiltrating normal cells in tumors, making the results less comparable to those obtained by RNase expression. MRP expression did not directly correlate with function in a calcein accumulation assay in 2 unselected cell lines. No gene amplification was observed by Southern-blot analysis, in the unselected cell lines or in tumor samples. In general, in cell lines, MRP gene expression was correlated with lower chemosensitivity to doxorubicin and etoposide, but not to cisplatin. However, MRP expression did not directly correlate with MRP function as assessed by a calcein accumulation assay in one of 2 unselected cell lines examined. Our results suggest that MRP may be implicated in drug resistance in unselected lung-cancer cell lines and its role in normal lung and primary lung cancer warrants further investigation in patients undergoing chemotherapy.

Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung.

UNLABELLED: DNA topoisomerases are ubiquitous nuclear enzymes, and important targets of cancer chemotherapy. Expression of topoisomerase genes is often correlated with in vitro chemosensitivity. We investigated the expression of the topoisomerase genes in normal lung and non-small cell lung cancer. Expression of topoisomerase II-alpha, topoisomerase II-beta, and topoisomerase I genes has been assessed in tumor samples of 60 patients who underwent operation for a non-small cell lung carcinoma, by RNase protection assay, and by immunohistochemistry. The expression of topoisomerase II-alpha gene was either undetectable or very low in normal lung, while most NSCLC expressed readily quantifiable levels of this gene. No alteration of the topoisomerase II-alpha gene was found by Southern blotting in the NSCLC samples. In contrast to topoisomerase II-alpha, topoisomerase II-beta was expressed in most normal as well as in tumor tissue samples, at a similar level. The levels of expression of both topoisomerase II isoforms was lower than that of human lung cancer cell lines. The results of the topoisomerase II mRNA expression were confirmed by immunohistochemistry. Whereas topoisomerase II-alpha staining was mainly limited to the nucleus, staining with topoisomerase II-beta antibody was exclusively observed in nucleoli. Topoisomerase I was localized in the nuclei and expression was mainly limited to tumor cells. By RNase protection, topoisomerase I expression in NSCLC samples was in the range of that of human lung cancer cell lines. The expression of the topoisomerase genes did not seem to be coordinated. In tumor cells, there was a positive association between expression of topoisomerase II-alpha and Ki-67, a marker of cell proliferation, as assessed by immunohistochemistry, but not with topoisomerase II-beta or topoisomerase I. Clinical characteristics of the patients, and their survival did not appear to be correlated to the level of expression of any of the topoisomerase genes, although a trend towards a shorter survival was observed in patients whose tumors expressed relatively high topoisomerase II-alpha mRNA levels. IN CONCLUSION: (1) the two isoforms of topoisomerase II are differentially expressed in normal lung and NSCLC cells; (2) higher topoisomerase II-alpha expression is associated with higher cell proliferation in NSCLC; (3) the expression of topoisomerase II-alpha and topoisomerase I, but not of topoisomerase II-beta, was higher in tumor cells compared to normal lung. Given the differential expression of topoisomerases in normal lung and tumors, research of more potent and specific topoisomerase inhibitors might prove beneficial in non-small cell lung cancer. Immunohistochemistry may be indicated in prospectively investigating the correlation between expression of topoisomerases and results of chemotherapy treatment.

Differential expression of the c-kit proto-oncogene in germ cell tumours.

The c-kit proto-oncogene product and its ligand stem cell factor play an important role in haematopoiesis, spermatogenesis, and melanogenesis. Using an anti-c-kit antiserum raised against a synthetic peptide, we studied the immunohistochemical expression of the c-kit gene product in 60 germ cell tumours (GCTs) (53 testicular, 7 extragonadal), derived from primary GCTs in 45 cases and metastatic tumours in 15 cases. Twenty-eight out of 28 seminomas showed c-kit membranous staining in the majority of cells. A similar pattern of expression was seen in intratubular germ cell neoplasia. Nine out of 29 (32 per cent) non-seminomas displayed cytoplasmic, but not membranous, c-kit immunoreactivity in occasional cells. In three mixed GCTs, c-kit expression was limited to the seminoma component. In normal testis, c-kit expression was observed in some basal tubular cells, corresponding to undifferentiated spermatogonia. These results suggest a role for c-kit in the oncogenesis of GCT, where down-regulation of c-kit might be a critical step during progression from seminomas to non-seminomas. Immunohistochemical analysis of c-kit should be considered as a diagnostic aid for GCT and in particular may be helpful in the identification of certain extragonadal seminomas.

Topoisomerase II alpha gene expression in childhood acute lymphoblastic leukemia.

Previously, we showed that in vitro resistance to daunorubicin (DNR) at initial diagnosis was related to a poor long-term clinical outcome in childhood acute lymphoblastic leukemia (ALL), and that cells of relapsed ALL were in vitro more resistant to DNR than cells of untreated ALL. Topoisomerase II (Topo II) is an intracellular target for anthracyclines and epipodophyllotoxins. Decreased levels and/or activity of Topo II have been associated with multidrug resistance in cell lines. We investigated Topo II alpha gene expression in fresh leukemic samples from 19 children with untreated and 14 children with relapsed ALL using a sensitive RNase protection assay. The in vitro cytotoxicity of the Topo II inhibitors DNA and teniposide (VM26) was measured using the MTT assay, and the cell cycle distribution of leukemic samples was analyzed by DNA flow cytometry. Results showed that (1) relapsed ALL samples were more resistant to DNR, but not to VM26 compared to untreated samples; (2) large interpatient variations existed in both Topo II alpha gene expression and in vitro cytotoxicity results; (3) Topo II alpha gene expression was detectable in 29/33 childhood ALL samples with a median expression of 5% the level of a relatively chemosensitive human small cell lung cancer cell line; (4) Topo II alpha gene expression did not differ between untreated and relapsed ALL; (5) Topo II alpha gene expression was positively correlated with the percentage of ALL cells in S- and G2M-phase, but not with the in vitro cytotoxicity of the drugs tested. In conclusion, resistance to DNR in childhood ALL can not be explained by decreased levels of Topo II alpha gene expression, but additional Topo II activity studies in fresh leukemia samples may need further exploration.

Effects of suramin on human lung cancer cell lines.

Suramin cytotoxicity was studied in a panel of human lung cancer cell lines by the MTT assay. The concentrations of suramin which induced 50% growth inhibition (IC50) ranged from 130 to 3715 microM for the cell lines growing in medium containing 10% fetal calf serum (FCS). In only one cell line was the IC50 at a concentration that can be reached in plasma of patients treated with suramin. Suramin was 18 and 3.3 times more cytotoxic on NCI-N417 cells growing in 2% FCS and in HITES serum-free medium, respectively, than growing in 10% FCS. No difference in suramin cytotoxicity was observed between small and non-small cell lung cancer cell lines. At the lower concentrations tested, suramin stimulated proliferation of the two small cell lung cancer cell lines, NCI-H187 and NCI-N417. Of several growth factors tested, none induced stimulation of growth in NCI-H187 and NCI-N417 cell lines, nor did they in any way alter the stimulatory effect of suramin. Cell counting, DNA flow cytometric analysis and Ki-67 staining confirmed a higher proliferative state in suramin-exposed NCI-H187 cells as compared with untreated cells. However, topoisomerase II-alpha gene expression remained unchanged, as assessed by northern blot analysis and immunostaining. Suramin had an inhibitory effect on topoisomerase II activity, as assessed by the kDNA decatenation assay, with an IC50 of approximately 40 microM. In conclusion, suramin has significant cytotoxic activity in a minority of human lung cancer cell lines, and it stimulates proliferation in some instances. The pleiotropic action of suramin observed should caution on the possibility of tumour acceleration in patients being treated with this drug.

Antiproliferative activity of the topoisomerase I inhibitors topotecan and camptothecin, on sub- and postconfluent tumor cell cultures.

We have assessed the antiproliferative effects of a 24-hr exposure to the topoisomerase I inhibitors, topotecan and camptothecin, on two colon and one ovarian human tumor cell lines, cultured as subconfluent and as multilayered postconfluent cultures. Chemosensitivity was measured by the sulforhodamine B assay. In general, postconfluent cultures were less sensitive to these agents, yielding GI50S (drug concentrations inhibiting growth by 50%) from 1.2 to more than 6000 times higher than those of subconfluent cultures. Both compounds displayed similar effects on subconfluent cells, inducing complete growth inhibition at concentrations ranging from 0.03 to 0.5 microM. Topotecan, however, was more potent than camptothecin in two out of the three cell lines tested as multilayered postconfluent cultures. Topoisomerase I mRNA expression on postconfluent cultures was 50% lower than on subconfluent cultures in the three cell lines studied. However, we did not detect any reproducible differences in topoisomerase I protein expression and in relaxation activity of supercoiled DNA between the two types of cultures. From accumulation experiments it appeared that the peak concentration of the lactone form of topotecan as well as the area under the concentration-time curve (AUC) were 2-fold higher in the monolayer than in the multilayer cultures. Therefore, the differences in the activity of topoisomerase I inhibitors under our experimental conditions were likely due to a decreased rate of proliferation of postconfluent cells, associated with a reduction in drug uptake.

Chemosensitivity to the indoloquinone EO9 is correlated with DT-diaphorase activity and its gene expression.

EO9, a new bioreductive indoloquinone alkylating agent, requires activation by a two-electron reduction, which can be catalysed by the NAD(P)H:quinone oxidoreductase DT-diaphorase (DTD) (EC 1.6.99.2). Seven human and four murine tumor cell lines from different histological origins were evaluated for their DTD enzyme activity (evaluated using dichlorophenolindophenol and EO9 as substrates), DTD gene expression and chemosensitivity to EO9. In general the cell lines could be divided into two groups: leukemic cells which were relatively resistant to EO9 (IC50 > or = 0.5 microM) and had no measurable DTD activity, and solid tumor cells, which were more sensitive to the drug (IC50 < 0.06 nM) and contained a high DTD activity (> 90 nmol/min/mg). The expression of the DTD gene was measured by semiquantitative PCR in the human cell lines and an excellent correlation between gene expression and enzyme activity was observed (r2 = 0.94). A higher DTD gene expression also correlated with higher chemosensitivity to EO9. Protection of chemosensitivity to EO9 by dicoumarol, a strong and specific inhibitor of DTD activity, was dependent on duration of exposure and concentration of dicoumarol. Inhibition was best observed by short exposure to dicoumarol and EO9 together, demonstrating that bioactivation of EO9 by DTD is essential. In conclusion, DTD activity and expression appear to predict sensitivity to EO9 in a variety of cell lines. Evaluation of activity or expression in patients' tumor samples might predict the response to EO9.

In vitro sequence-dependent synergistic effect of suramin and camptothecin.

Suramin, a hexasulphonated naphthylurea with activity in prostatic cancer, possesses a wide variety of antitumour mechanisms of action, one of which is the inhibition of topoisomerase II. In this in vitro study, suramin was combined with the topoisomerase I inhibitor, camptothecin. Several suramin concentrations (0.2-3000 microM) were combined with camptothecin (0.4 pM-20 microM) in MCF-7 and PC3 human cancer cell line cultures. In addition, we studied the topoisomerase II and I gene expression by northern blot analysis, and the cell cycle distribution by flow cytometry, after exposure to suramin. While there was only an additive effect when suramin and camptothecin were added simultaneously, a remarkable synergism was obtained when camptothecin was added after a 3-day exposure to suramin. Topoisomerase II and I gene expression and the number of cells in S phase were significantly reduced after exposure to suramin. In conclusion, interaction of suramin with camptothecin is schedule-dependent and can be synergistic. These findings might help in identifying optimal combinations of suramin or other topoisomerase II inhibitors, with topoisomerase I inhibitors.

Transformation of mouse fibroblasts with the oncogenes H-ras OR trk is associated with pronounced changes in drug sensitivity and metabolism.

Malignant activation of oncogenes ras or trk is implicated in a number of solid tumors and leukemias. We determined the chemosensitivity profile of wild-type mouse NIH-3T3 fibroblasts, and that of NIH-3T3 lines transformed by the H-ras (S2-721) and trk (106-632) oncogenes, against 11 different drugs from various classes. Differences in sensitivity were related to drug accumulation and metabolism. Both ras- and trk-transformed cell lines were less sensitive to cisplatin (CDDP) and doxorubicin (DXR) than the wild type. NIH-3T3 transformants expressing H-ras were less sensitive than those expressing trk or the wild type to the indoloquinone EO9, methotrexate and arabino-furanosylcytosine. No clear difference in sensitivity was observed for vincristine, VP-16, or the new cytidine analog 2',2'-difluoro-deoxycytidine. In both ras- and trk-transformed cell lines sensitivity to 5FU was increased moderately, but sensitivity to 5'deoxy-5-fluorouridine (5'dFUR) was increased markedly. Only the trk-transformed line NIH-3T3 was more sensitive to 2'deoxy-5-fluorouridine. Expression of P-glycoprotein was not different between the 3 cell lines but DXR accumulation in both mutants was decreased, indicating a non-P-glycoprotein-associated difference in sensitivity. Conversion of 5'dFUR to 5FU (catalyzed by pyrimidine nucleoside phosphorylases) was 5-10 times higher in both mutants than in the wild type. The activity of the phosphoribosyl-transferase (direct conversion of 5FU to FUMP) was comparable, but the rate of conversion of 5FU to fluorouridine (FUR) was lower in the wild type, as well as that of 5FU to FUMP via FUR. In contrast, the activity of thymidylate synthase, the target enzyme for fluoropyrimidines, was higher in the wild-type cells. The concentrations of both purine and pyrimidine nucleotides were lower in cells expressing trk. In conclusion, transformation of cells with the H-ras or trk oncogenes can markedly influence sensitivity to several drugs and affect normal metabolism and that of several anti-cancer agents.

Cytotoxic effects of anticancer agents on subconfluent and multilayered postconfluent cultures.

The cytotoxic effects of conventional (doxorubicin, 5-fluorouracil, cisplatin) and investigational (2',2'-difluorodeoxycytidine, hexadecylphosphocholine, EO9, rhizoxin) anticancer drugs were studied in subconfluent and multilayered postconfluent cultures of human colon and ovarian carcinoma cell lines. Chemosensitivity was assessed 4 days after a 24-h drug exposure with the sulphorhodamine B assay. Except for rhizoxin, all drugs tested yielded an EC50 (drug concentration producing absorbance readings 50% lower than those of non-treated wells) in postconfluent cultures that were higher than an EC50 obtained with subconfluent cultures. Compared with subconfluent cultures, postconfluent cultures showed decreased cellular nucleotide concentrations and ATP/ADP ratios, in addition to an increased percentage of G0/G1 cells. The activity of DT-diaphorase, a reductase involved in the bioactivation of EO9, was similar in sub- and postconfluent cultures. These results indicate similarity of the postconfluent model presented with those obtained with in vivo models and more complex in vitro techniques.