Microenvironment involved in FPR1 expression by human glioblastomas.

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.

Targeting breast cancer through its microenvironment: current status of preclinical and clinical research in finding relevant targets.

It is increasingly evident that not only breast cancer cells, but also the tissue embedding these cells: the tumor microenvironment, plays an important role in tumor progression, metastasis formation and treatment sensitivity. This review focuses on the current knowledge of processes by which the microenvironment affects breast cancer, including formation of the metastatic niche, metabolic stimulation, stimulation of tumor cell migration, immune modulation, angiogenesis and matrix remodeling. The number of drugs targeting key factors in these processes is expanding, and the available clinical data is increasing. Therefore current strategies for intervention and prediction of treatment response are outlined. At present, targeting the formation of the metastatic niche and metabolic stimulation by the breast cancer microenvironment, are already showing clinical efficacy. Intervening in the stimulation of tumor cell migration and immune modulation by the microenvironment upcoming fields of great research interest. In contrast, targeting microenvironmental angiogenesis or matrix remodeling appears to be of limited clinical relevance in breast cancer treatment so far. Further research is warranted to optimize intervention strategies and develop predictive tests for the relevance of targeting involved factors within the microenvironment in order to optimally personalize breast cancer treatment.

Unravelling mechanisms of cisplatin sensitivity and resistance in testicular cancer.

Testicular cancer is the most frequent solid malignant tumour type in men 20-40 years of age. At the time of diagnosis up to 50% of the patients suffer from metastatic disease. In contrast to most other metastatic solid tumours, the majority of metastatic testicular cancer patients can be cured with highly effective cisplatin-based chemotherapy. This review aims to summarise the current knowledge on response to chemotherapy and the biological basis of cisplatin-induced apoptosis in testicular cancer. The frequent presence of wild-type TP53 and the low levels of p53 in complex with the p53 negative feed-back regulator MDM2 contribute to cisplatin sensitivity. Moreover, the high levels of the pluripotency regulator Oct4 and as a consequence of Oct4 expression high levels of miR-17/106b seed family and pro-apoptotic Noxa and the low levels of cytoplasmic p21 (WAF1/Cip1) appear to be causative for the exquisite sensitivity to cisplatin-based therapy of testicular cancer. However, resistance of testicular cancer to cisplatin-based therapy does occur and can be mediated through aberrant levels of the above mentioned key players. Drugs targeting these key players showed, at least pre-clinically, a sensitising effect to cisplatin treatment. Further clinical development of such treatment strategies will lead to new treatment options for platinum-resistant testicular cancers.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway.

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2-p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.

The chemokine network, a newly discovered target in high grade gliomas.

Chemokines are small cytokines, characterised by their ability to induce directional migration of cells by binding to chemokine receptors. They are known to play a role in tumour development, angiogenesis and metastasis. Interestingly, the chemokine network also contributes to the progression of gliomas, mainly by intensifying their characteristic invasive character. The main hurdle in treatment of these tumours is their infiltration of surrounding tissues, hampering complete surgical tumour removal. Standard postsurgical treatment with radio- and chemotherapy is of limited effect. Therefore drugs that target the chemokine system in high grade gliomas might fill the gap existing in the current approach. This review presents the current knowledge of the role of chemokine network in the development of the central nervous system, in brain physiology and the involvement in brain tumour progression. Finally, current studies exploring new compounds targeting the chemokine network in cancer patient are discussed.

Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation.

The aim of this study was to find factors that could explain the accumulation difference of mitoxantrone in the BCRP1-negative GLC4-MITO cell line compared to GLC4. Comparative genomic hybridisation (CGH) was applied to determine chromosomal differences between GLC4 and GLC4-MITO. Comparative genomic hybridisation analysis revealed gain of 2q, 6p, 9q, 13q, 14q, 15q, 19q and Xp and loss of 1p, 2q, 3p, 3q, 4q, 6q, 8q, 11p, 16p, 17q, 18p, 20p and Xq. In the over-represented chromosomal areas, seven transporter genes were identified: ABCB6, ABCB2 (TAP1), ABCB3 (TAP2), ABCF1 (ABC50), ABCC10 (MRP7), ABCA2 (ABC2) and ABCC4 (MRP4). No RNA or protein upregulation was observed for ABCB6, ABCF1, ABCC10, ABCC4, ABCB2 and ABCB3, but an increased expression was detected for ABCA2 mRNA in GLC4-MITO. ABCA2 is known to be involved in resistance to estramustine. In the MTT assay, GLC4-MITO was two-fold resistant to estramustine compared to GLC4. Coincubation with estramustine and mitoxantrone increased mitoxantrone accumulation in GLC4-MITO, while this was not affected in GLC4. This suggests that estramustine is able to block mitoxantrone efflux in GLC4-MITO cells. These data reveal that cellular reduction of mitoxantrone in a mitoxantrone-resistant cell line is associated with overexpression of ABCA2.

An in vitro model for purging of tumour cells from ovarian tissue.

BACKGROUND: Cryopreservation and autografting of ovarian tissue may preserve fertility after cancer treatment, but could be hampered by tumour cell contamination. Epithelial tumour cell lysis can be obtained with cytotoxic T cell retargeting through the bispecific antibody BIS-1, with combined affinity for the T-cell receptor and epithelial glycoprotein-2 (EGP-2). Our aim was to study the concept of tumour cell purging in the setting of a suspension of ovarian tissue. METHODS: Human ovarian tissue was brought into suspension by mechanical and enzymatic treatment. Cells of the MCF-7 breast cancer cell line and activated human lymphocytes were co- incubated for 4 h with or without BIS-1 and with or without ovarian suspension. After incubation, MCF-7 cell death and cell growth were evaluated by fluorescent cell detection and MTT assay, respectively. Ovarian tissue morphology was evaluated immunohistochemically. RESULTS: MCF-7 cell death and cell growth inhibition increased with increasing ratios of lymphocytes to MCF-7 cells. BIS-1 addition gave further augmentation, with a maximum depletion of growing MCF-7 cells of 89% (SD 11%) versus without BIS-1, 23% (SD 15%; P < 0.001). Follicles remained morphologically intact. CONCLUSIONS: Purging of added epithelial tumour cells from ovarian tissue with BIS-1 is possible in vitro. Morphologically, follicles remain intact after this procedure. This method may contribute to safer replacement of ovarian tissue in female cancer survivors.

Cytotoxicity of rhein, the active metabolite of sennoside laxatives, is reduced by multidrug resistance-associated protein 1.

Anthranoid laxatives, belonging to the anthraquinones as do anthracyclines, possibly increase colorectal cancer risk. Anthracyclines interfere with topoisomerase II, intercalate DNA and are substrates for P-glycoprotein and multidrug resistance-associated protein 1. P-glycoprotein and multidrug resistance-associated protein 1 protect colonic epithelial cells against xenobiotics. The aim of this study was to analyse the interference of anthranoids with these natural defence mechanisms and the direct cytotoxicity of anthranoids in cancer cell lines expressing these mechanisms in varying combinations. A cytotoxicity profile of rhein, aloe emodin and danthron was established in related cell lines exhibiting different levels of topoisomerases, multidrug resistance-associated protein 1 and P-glycoprotein. Interaction of rhein with multidrug resistance-associated protein 1 was studied by carboxy fluorescein efflux and direct cytotoxicity by apoptosis induction. Rhein was less cytotoxic in the multidrug resistance-associated protein 1 overexpressing GLC4/ADR cell line compared to GLC4. Multidrug resistance-associated protein 1 inhibition with MK571 increased rhein cytotoxicity. Carboxy fluorescein efflux was blocked by rhein. No P-glycoprotein dependent rhein efflux was observed, nor was topoisomerase II responsible for reduced toxicity. Rhein induced apoptosis but did not intercalate DNA. Aloe emodin and danthron were no substrates for MDR mechanisms. Rhein is a substrate for multidrug resistance-associated protein 1 and induces apoptosis. It could therefore render the colonic epithelium sensitive to cytotoxic agents, apart from being toxic in itself.

Uptake mechanisms of L-3-[125I]iodo-alpha-methyl-tyrosine in a human small-cell lung cancer cell line: comparison with L-1.

The radiolabelled amino acid analogue L-3-[125I]iodo-alpha-methyl-tyrosine (IMT) is under evaluation in brain tumours, where it reflects amino acid transport activity, but is also taken up in many other tumour types. This study investigated the uptake mechanism of IMT in tumour cells not derived from brain tumours, in comparison with the native amino acid 14C-tyrosine (Tyr) from which IMT is derived. Human GLC4 small-cell lung cancer cells in log-phase were incubated with IMT and Tyr. Tracer uptake was determined in various buffers, incubation periods, concentrations of specific amino acid transport blockers, pH and temperature. IMT uptake was very fast, reaching a plateau within 5 min, while Tyr kept on accumulating for > 60 min. Based on steady-state experiments, > 90% of IMT uptake could be attributed to amino acid transport activity. The L transport system was the most important, both for IMT and Tyr. IMT uptake into GLC4 tumour cells is almost completely the result of amino acid transport activity (especially the L system) and is very similar to Tyr uptake. Therefore, also outside the brain, IMT is a metabolic tracer that may reflect the increased amino acid transport that is characteristic for malignant tumours.

Effect of specific or random c-DNA priming on sensitivity of tyrosinase nested RT-PCR: potential clinical relevance.

The reverse transcriptase polymerase chain reaction (RT-PCR) can be of clinical relevance in identifying malignant melanoma cells in blood or tissues of patients at risk for disseminated melanoma. The diagnostic value of this marker however, is still controversial. The objective of this study was to compare and quantify the difference in sensitivity of the nested RT-PCR for tyrosinase, with respect to the method utilized to produce the template c-DNA. We found a difference of a factor 10 in favor of a specific priming versus a random one. We concluded that this difference can be exploited in the analysis of blood samples. However, in the analysis of lymph node specimens, where the chance of positivity due to tyrosinase positive non-melanoma cells is much higher, the choice of a highly sensitive assay should be made with caution.

Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes.

The specificity and sensitivity of the nested reverse transcriptase polymerase chain reaction (RT-PCR) on tyrosinase was studied, for the detection of micrometastases of malignant melanoma. The specificity was assessed in the blood of six healthy donors, four patients with non-melanoma cancers of which one patient was treated with granulocyte-colony stimulating factor. Lymph nodes of nine patients without malignant melanoma were tested and four cell lines of various other tumours. Six of the nine non-melanoma lymph nodes were positive in this assay. The sensitivity was tested in a spike experiment in vitro, using a melanoma cell line. The detection limit was ten melanoma cells per 10(7) peripheral blood lymphocytes.

Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity.

Deletion of the multidrug resistance gene MRP1 has been demonstrated in acute myeloid leukemia (AML) patients with inversion of chromosome 16 (inv[16]). These AML patients are known to have a relatively favorable prognosis, which suggests that MRP1 might play an important role in determining clinical outcome. This study analyzed MRP1 deletion by fluorescent in situ hybridization (FISH), with a focus on inv(16) AML patients. Functional activity of multidrug resistance protein (MRP) was studied in a flow cytometric assay with the use of the MRP substrate carboxyfluorescein (CF) and the inhibitor MK-571. MRP1, MRP2, and MRP6 messenger RNA (mRNA) expression was determined with reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with normal bone marrow cells. MRP1 deletion was detected in 7 AML patients; 2 cases showed no MRP1 FISH signals, and 5 cases had 1 MRP1 signal, whereas in 4 AML patients with inv(16) no MRP1 deletions were observed. A variability in MRP activity, expressed as CF efflux-blocking by MK-571, was observed (efflux-blocking factors varied between 1.2 and 3.6); this correlated with the number of MRP1 genes (r = 0.91, P <. 01). MRP activity in the AML cases was not different from normal hematopoietic cells. MRP1 mRNA was detected in patients with 1 or 2 MRP1 FISH signals, but not in patients with no MRP1 signals. MRP2 and MRP6 mRNA were expressed predominantly in AML samples with 1 MRP1 signal, whereas in normal bone marrow cells no MRP2 and MRP6 mRNA was observed. In conclusion, this study shows that MRP activity varies among inv(16) AML cases and does not differ from that in normal hematopoietic cells; this might be in part due to the up-regulation of other MRP genes.

Phase I study of transforming growth factor-beta3 mouthwashes for prevention of chemotherapy-induced mucositis.

The purpose of this study was to establish the safety and tolerability of recombinant transforming growth factor-beta3 (TGF-beta3; CGP 46614) mouthwashes intended for prevention of chemotherapy-induced mucositis. Local effects were especially analyzed by objective and subjective measurements of mucositis. Secondary aims were analysis of potential systemic exposure and development of anti-TGF-beta3-antibodies. Eleven breast cancer patients received chemotherapy with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. (n = 8) or 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days (n = 3). TGF-beta3 mouthwashes (10 ml; provided by Novartis, Basel, Switzerland) were administered for 4 days, four times a day, starting 1 day before chemotherapy. The dose was escalated in following patients from 25 microg/ml (n = 3) to 50 microg/ml (n = 3) and 100 microg/ml (n = 5). Clinically, the mucosa was scored objectively and according to WHO criteria. The percentage of viable oral epithelial cells was determined by trypan blue dye exclusion. Morphology of cells was assessed in buccal smears. Plasma samples were collected for determination of TGF-beta3 levels and anti-TGF-beta3-antibodies. Adverse events were recorded by the patient in a diary. Mouthwashes with TGF-beta3 were well tolerated. Three patients scored for mucositis > grade 0 (WHO grading criteria). The percentage of viable oral epithelial cells in patients treated with 1.5 g/m2 cyclophosphamide i.v., 80 mg/m2 epirubicin i.v., and 1.0 g/m2 5-fluorouracil i.v. was stable, whereas in patients treated with 1.6 g/m2 carboplatin i.v., 480 mg/m2 thiotepa i.v., and 6 g/m2 cyclophosphamide i.v. divided over 4 days, an increase was observed. The morphology of buccal cells showed a transient shift from mature to immature cells in the first week. Neither systemic absorption of TGF-beta3 nor development of TGF-beta3-antibodies was observed. TGF-beta3 mouthwashes were well tolerated and deserve further study in preventing chemotherapy-induced mucositis.

Combined cytotoxic effects of tumor necrosis factor-alpha with various cytotoxic agents in tumor cell lines that are drug resistant due to mutated p53.

Several studies suggest that tumor necrosis factor-alpha (TNF) is able to overcome drug resistance in tumors. Whether TNF is able to do so in tumor cell lines that are drug resistant due to a mutation in the tumor suppressor gene p53 is unclear. Therefore, we studied the in vitro cytotoxic effects of TNF combined with various cytotoxic agents in a model consisting of a human ovarian cancer cell line containing endogenous wild-type p53 (wtp53) and sublines that were made drug resistant against various cytotoxic agents by transfection of several forms of mutated p53 (mtp53). Using the microculture tetrazolium assay, the cytotoxic effects of TNF alone, the cytotoxic agents VM-26, melphalan, cisplatin, vinblastine, paclitaxel, and mitoxantrone, plus the combined effects of 10 ng/ml TNF added 30 min before various concentrations of the cytotoxic agents were established. Compared with the control cell line (A2780/cmv), two cell lines transfected with mtp53 (A2780/m248 and A2780/m273) showed increased resistance against several cytotoxic agents but also an enhanced sensitivity to TNF. Interaction of TNF with the cytotoxic drugs was additive in the drug-sensitive control cell line as well as in the drug-resistant sublines. However, because of the increased sensitivity of A2780/m248 to TNF at the dose used for the combinations, the combination of TNF with several cytotoxic drugs reduced the level of resistance in A2780/m248 compared with the control cell line A2780/cmv. In conclusion, this study shows that addition of TNF can ameliorate resistance to cytotoxic agents in a subline that is drug-resistant because of mutated p53. This reduction in resistance by TNF is not due to synergistic interaction, but to collateral sensitivity to TNF.

Enhanced sensitivity to tumor necrosis factor-alpha in doxorubicin-resistant tumor cell lines due to down-regulated c-erbB2.

We have studied the relationship between tumor necrosis factor (TNF)-sensitivity and doxorubicin-resistance in our doxorubicin-resistant cell line panel consisting of the parental cell line GLC4 plus GLC4-Adr2x and GLC4-Adr350x with respective resistance factors of 2 and 350 compared with GLC4. At the highest dose of 1000 ng/ml TNF, GLC4 was almost completely resistant to TNF, whereas 37% and 68% growth inhibition was observed in GLC4-Adr2x and GLC4-Adr350x, respectively. Sensitivity to TNF appeared to correlate inversely with the expression and gene copies of topoisomerase IIalpha in these cell lines. The gene encoding for c-erbB2 is in the proximity of the topoisomerase IIalpha gene and its product is a known cause for TNF-resistance. We found that our doxorubicin-resistant cell lines with decreased topoisomerase IIalpha gene copies have a simultaneous decrease in c-erbB2 gene copies, probably due to linkage between these 2 genes. This reduced number of c-erbB2 gene copies resulted in decreased c-erbB2 expression and subsequently in increased sensitivity to TNF. Additionally, we tried to establish some of the mechanisms associated with TNF-resistance in GLC4 related to c-erbB2 overexpression. There was no difference in TNF-receptor-1 expression between the cell lines. Compared with the TNF-sensitive GLC4-Adr350x, GLC4 appeared to have a decreased activation of NF-kappaB after exposure to TNF that might indicate a reduced TNF-receptor function. In GLC4, increased Bcl-2 expression was found, a protein described to confer TNF-resistance. Moreover, it was demonstrated that combining TNF with the poly-ADP-ribose polymerase (PARP) inhibitors 6-aminonicotinamide and 3-aminobenzamide did not affect TNF-sensitivity in GLC4 and GLC4-Adr350x, excluding a pivotal role of PARP in TNF-resistance in these cell lines. In conclusion, our data show that doxorubicin-resistant tumor cell lines with decreased topoisomerase IIalpha gene copies can become sensitive to TNF due to loss of c-erbB2 gene copies. We also found that several mechanisms associated with c-erbB2 overexpressing contribute to TNF-resistance in GLC4.

Differential effects of all-trans-retinoic acid, docosahexaenoic acid, and hexadecylphosphocholine on cisplatin-induced cytotoxicity and apoptosis in a cisplantin-sensitive and resistant human embryonal carcinoma cell line.

Apart from modulation of tumor-cell drug sensitivity, induction of differentiation might be an alternative in the treatment of tumors resistant to cytotoxic drugs. In this report the capacity to induce differentiation and to modulate the cis-diamminedichloroplatinum(II) (CDDP) sensitivity of all-trans-retinoic acid (RA), docosahexaenoic acid (DCHA), and hexadecylphosphocholine (HePC) is examined in human germ-cell tumor cell lines. In the embryonal carcinoma cell line Tera-2 and its 3.7-fold CDDP-resistant subline Tera2-CP, we evaluated the effects of 96 h of pretreatment with RA (0.1 microM), DCHA (23 microM), and HePC (25 microM) on differentiation induction and on CDDP-induced cytotoxicity, DNA platination (4-h incubation), and apoptosis (continuous incubation). Without drug treatment, Tera2-CP showed less apoptosis than Tera-2. Pretreatment with RA decreased the cytotoxicity and apoptosis induced by CDDP without resulting in decreased DNA platination and increased differentiation in both cell lines. DCHA enhanced CDDP-induced cytotoxicity and apoptosis and did not affect the embryonal character of either cell line. HePC did not affect CDDP cytotoxicity or differentiation in either cell lines. Effects of the modulators on differentiation and on CDDP-induced cytotoxicity, DNA platination, and apoptosis did not differ between Tera-2 and Tera2-CP. RA can be applied for differentiation induction in CDDP-resistant germ-cell tumor models. However, in this model, RA reduced the apoptotic susceptibility. DCHA potentiated CDDP cytotoxicity in vitro; its in vivo modulatory capacity in germ-cell tumor cells deserves further study.

Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin-resistant tumor cell lines.

Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines. Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC4), its 300-fold doxorubicin-resistant and multidrug resistance-associated protein (MRP)-over-expressing subline (GLC4/ADR), an ovarian carcinoma cell line (A2780) and its 100-fold doxorubicin resistant and P-glycoprotein (P-gp)-overexpressing subline A2780AD. Cross-resistance, measured with the MTT assay at MMRDX concentration resulting in 50% growth inhibition, was 1.8-fold in GLC4/ADR and 4.5-fold in A2780AD compared to their respective parental cell lines. Cellular MMRDX accumulation was equal in GLC4 and GLC4/ADR and 2-fold lower in A2780AD compared to A2780. Doxorubicin fluorescence was analyzed with confocal laser scan microscopy. Fluorescence was nuclear in sensitive, and cytoplasmic in resistant, cell lines, while MMRDX fluorescence was found in the nucleus in all cell lines. Pre-incubation with the MRP blocker MK 571 restored in GLC4/ADR cells the nuclear doxorubicin fluorescence pattern, as observed in GLC4 cells. MMRDX, thus, can largely overcome cross-resistance in these P-gp- and MRP-overexpressing doxorubicin-resistant cell lines. Our results suggest that MMRDX is not a substrate for MRP-mediated resistance.

A new in vitro assay for quantitation of chemotherapy-induced mucositis.

Patients receiving high-dose chemotherapy (HD-CT) are at risk of severe mucositis. Most prevention studies evaluate the degree of mucositis on clinical, and therefore subjective, measurements. The aim of this study was to develop an objective in vitro assay of chemotherapy-induced mucositis. Twelve patients with locally advanced breast carcinoma received HD-CT followed by peripheral stem cell reinfusion. Before and twice weekly after HD-CT, the mucosa was evaluated by an oral washing, a buccal smear and the World Health Organization (WHO) toxicity grading; furthermore, blood leucocyte levels were determined. For the oral washings, the percentage of viable epithelial cells was determined by trypan blue dye exclusion and leucocytes were counted by fluorescence microscopy after incubation with acridine orange. Maturity of buccal cells was assessed by staining buccal smears for morphology according to Papanicolaou (Whitacker D and Williams V, 1994). Eight healthy volunteers served as controls. The mean percentage (+/- s.e.m.) of viable oral epithelial cells was stable in controls (44 +/- 2%). In patients, they increased after HD-CT, which was significant after day 7 compared with pretreatment (P < or = 0.05). In addition, a shift from mature to immature epithelial cells in buccal smears was observed. Oral leucocyte levels were closely correlated with the blood leucocyte counts. The WHO score followed the results of these other evaluations with some delay. The viability of buccal cells obtained by oral washings increases after HD-CT. This is possibly because of desquamation of the upper oral mucosa layer, with a shift from mature to more immature cells. These data can be quantitated, and this assay may therefore be useful in studies aimed at prevention of mucositis.

Cellular basis for differential sensitivity to cisplatin in human germ cell tumour and colon carcinoma cell lines.

Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels of the cell lines correlated with CDDP concentrations inhibiting cell survival by 50% (IC50); total cellular sulphydryl content (TSH) was unexpectedly inversely correlated with IC50. IC50 correlated neither with glutathione S-transferase (GST) nor with GST pi expression, topoisomerase I or II activity. Immediately after 4 h incubation with CDDP, platinum (Pt) accumulation and Pt bound to DNA were not correlated, but after another 24 h drug-free culture, Pt binding to DNA in germ cell tumour but not in colon carcinoma cell lines correlated with IC50. With the exception of in vitro sensitivity and TSH, none of the parameters studied discriminated between the two groups of cell lines. Correction of CDDP sensitivity parameters for phenotypical differences did not influence statistical correlations. Analysis of variance revealed a correlation between IC50 and the combination of glutathione, GST activity and Pt bound to DNA. But at other CDDP cytotoxicity levels sensitivity was also correlated with Pt accumulation, topoisomerase II activity and TSH in various combinations. This model of intrinsic CDDP resistance showed that multiple parameters ought to be studied to explain CDDP resistance, but did not elucidate the cause of the unique sensitivity of germ cell carcinoma, although the unexpected values of TSH deserve further attention.