Coby Meijer

Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research.

Endothelial cells (EC) are currently used as in vitro model systems for various physiological and pathological processes, especially in angiogenesis research. Primary EC have a limited lifespan and display characteristics that differ from batch to batch due to their multidonor origin. In recent years many groups have established EC lines. This Review gives an overview of the advantages and disadvantages of currently available vascular EC lines. Its aim is to help the investigator to decide which cell line matches his or her research goal best. Truly immortalized cell lines are generally better characterized and more stable in their endothelial traits than EC that were given an extended life span. Presently the best characterized macro- and micro-vascular EC lines are EA.hy926 and HMEC-1, respectively.

Platelets and granulocytes, in particular the neutrophils, form important compartments for circulating vascular endothelial growth factor.

The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer (n= 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 μg VEGF/l) compared with the healthy volunteers (164 μg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.

99mTc-sestamibi is a substrate for P-glycoprotein and the multidrug resistance-associated protein.

99mTc-sestamibi (99mTc-MIBI) is a substrate for the P-glycoprotein (P-gp) pump but it is not known whether it is a substrate for the multidrug resistance-associated protein (MRP) pump. Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP. 99mTc-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1. 99mTc-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone. 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines. Glutathione depletion elevated 99mTc-MIBI concentration in GLC4/ADR150x. Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR150x vs GLC4. 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR150x. These results show that 99mTc-MIBI is involved in MRP-mediated efflux. The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this gamma-rays-emitting complex is tested for tumour efflux measurements.

The insulin-like growth factor system and sarcomas†

Sarcomas are a diverse group of malignant mesenchymal tumours arising from bone and soft tissues. The identification of critical cellular signalling pathways in sarcomas is an important issue for the development of new targeted therapies. This review highlights the experimental and clinical evidence supporting the role of the insulin‐like growth factor (IGF) signalling system in the cellular transformation and progression of several types of sarcoma, including rhabdomyosarcoma, synovial sarcoma, leiomyosarcoma, Ewings sarcoma and osteosarcoma. Preclinical data suggest that the IGF system could be a promising target for therapy in these sarcomas. Currently, therapies interrupting IGF signalling have been or are being developed. In recent phase 1 clinical studies with humanized monoclonal antibodies directed against IGF receptor type 1 (IGF‐1R), objective tumour responses were observed in several patients with Ewings sarcoma, encouraging further clinical testing in Ewings sarcoma and other sarcoma (sub)types. Moreover, the occasional occurrence of paraneoplastic hypoglycaemia as a result of the secretion of incompletely processed forms of pro‐IGF‐II by sarcomas is discussed. Copyright

Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug‐resistance protein 1

Intracellular glutathione‐conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300× doxorubicin‐resistant, MRP1‐over‐expressing, GLC4‐Adr subline. Transport of non‐toxic concentrations of monochlorobimane and 5‐chloro‐methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4‐Adr. Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1‐chloro‐2,4‐dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans‐Golgi network. In GLC4‐Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP1 functions as a glutathione‐conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles. Int. J. Cancer 76:55–62, 1998.© 1998 Wiley‐Liss, Inc.

JM216-, JM118-, and cisplatin-induced cytotoxicity in relation to platinum-DNA adduct formation, glutathione levels and p53 status in human tumour cell lines with different sensitivities to cisplatin

The aim of this study is to establish anti-tumour potency of the new oral platinum drug JM216 and its metabolite JM118 in relation to the platinum (Pt)-DNA adduct formation, glutathione (GSH)-levels, and p53 status in human cancer cell lines with different sensitivities to cisplatin (CDDP). These parameters were studied in the CDDP sensitive human germ cell cancer cell line Tera and the small-cell lung cancer cell line GLC4 and their sublines with in vitro acquired CDDP resistance, Tera-CP and GLC4-CDDP, in a human ovarian cancer cell line transfected with mutant p53 (A2780/mt273) and with an empty vector as control (A2780/cmv), and in the intrinsic CDDP resistant human non-small-cell lung cancer cell line SW1573/S1 and colon carcinoma cell line Caco-2. Cytotoxicity was tested with the microculture tetrazolium (MTT)-assay. Pt-DNA adduct levels were assessed immunocytochemically. Quantitative analysis was performed by double fluorescence video microscopy. Results were correlated with GSH levels and p53 status of the cell lines. This study showed that both JM216 and JM118 can partially circumvent intrinsic and acquired resistance to CDDP. Drug-induced cytotoxicity only correlated negatively with GSH levels for JM216 and CDDP in the tested unselected cell lines. At equimolar basis, JM216 induced lower levels of Pt-DNA adducts in the various cell lines than JM118 and CDDP, whereas the JM118-induced amount and pattern of Pt-DNA adducts was comparable to CDDP. No difference in initial Pt-DNA adducts levels was observed between cell lines sensitive, acquired or intrinsic resistant to CDDP suggesting a Pt-resistance mechanism based on tolerance or increased repair, rather than decreased initial Pt-DNA adduct formation.

Variation in Bleomycin Hydrolase Gene Is Associated With Reduced Survival After Chemotherapy for Testicular Germ Cell Cancer

PURPOSEnResponse to chemotherapy may be determined by gene polymorphisms involved in metabolism of cytotoxic drugs. A plausible candidate is the gene for bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, an essential component of chemotherapy regimens for disseminated testicular germ-cell cancer (TC). We investigated whether the single nucleotide polymorphism (SNP) A1450G of the BLMH gene (rs1050565) is associated with survival.nnnPATIENTS AND METHODSnData were collected on survival and BLMH genotype of 304 patients with TC treated with bleomycin-containing chemotherapy at the University Medical Center Groningen, the Netherlands, between 1977 and 2003. Survival according to genotype was analyzed using Kaplan-Meier curves with log-rank testing and Cox regression analysis with adjustment for confounders.nnnRESULTSnBLMH gene SNP A1450G has a significant effect on TC-related survival (log-rank P = .001). The homozygous variant (G/G) genotype (n = 31) is associated with decreased TC related survival compared with the heterozygous variant (A/G; n = 133) and the wild-type (A/A; n = 140). With Cox regression the G/G genotype proves to be an unfavorable prognostic factor, in addition to the commonly used International Germ Cell Consensus Classification prognosis group, with a hazard ratio of 4.97 (95% CI, 2.17 to 11.39) for TC-related death. Furthermore, the G/G genotype shows a higher prevalence of early relapses.nnnCONCLUSIONnThe homozygous variant G/G of BLMH gene SNP A1450G is associated with reduced survival and higher prevalence of early relapses in TC patients treated with bleomycin-containing chemotherapy. This association is hypothesis generating and may eventually be of value for risk classification and selection for alternative treatment strategies in patients with disseminated TC.

In vivo imaging of hepatobiliary transport function mediated by multidrug resistance associated protein and P-glycoprotein

Multidrug resistance associated proteins (MRPs) and P-glycoprotein (P-gp) are involved in hepatobiliary transport of various compounds. Our aim was (1) to define transporter specificity of the cholescintigraphic agents 99mTc-HIDA and 99mTc-MIBI, which are used clinically for myocardial perfusion measurements; and (2) to deduce MRP and P-gp functions in vivo from hepatic 99mTc kinetics. Accumulation of radioactivity was measured in the human tumor cell lines GLC4, GLC4/ADR150x (MRP1-overexpressing/P-gp-negative) and GLC4/P-gp (P-gp-overexpressing). Bile secretion was quantified in untreated and in glutathione-depleted control and MRP2-deficient (GY/TR−) rats. Hepatobiliary transport was measured using a gamma camera in both types of rats. 99mTc-HIDA accumulated 5.8-fold less in GLC4/ADR150x calls than in GLC4 or GLC4/P-gp cells. In GLC4/ADR150x, the cellular 99mTc-HIDA content was increased 3.4-fold by the MRP1,2 inhibitor MK571 (50xa0μM), while MK571 had no measurable effect in GLC4 and GLC4/P-gp cells. 99mTc-MIBI accumulated less in GLC4/P-gp and GLC4/ADR150x cells than in GLC4 cells. Bile secretion of 99mTc-HIDA was impaired in GY/TR− compared to control rats and not affected by glutathione depletion in GY/TR− rats. Hepatic secretion of 99mTc-HIDA was slower in GY/TR− (t1/2 40xa0min) than in control rats (t1/2 7xa0min). Bile secretion of 99mTc-MIBI was similar in both rat strains and impaired by glutathione depletion in control rats only, indicating compensatory activity of additional transporter(s) in GY/TR− rats. 99mTc-HIDA is transported only by MRP1,2 only, while 99mTc-MIBI is transported by P-gp and MRP1,2. The results indicate that hepatic P-gp and MRP1,2 function can be assessed in vivo by sequential use of both radiopharmaceuticals.

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells.

It has been suggested that the expression of certain heat‐shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ‐cell tumor cell lines, 1 human small‐cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ‐cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity, i.e. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC4) and its subline with acquired resistance (GLC4‐cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC4 cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis.

Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric focussing. Both GST activity and GST pi amount were not related to histopathologic type, differentiation grade, or tumour volume index in untreated malignant tumours. GST isoenzyme patterns were identical in benign tumours and malignant tumours before and after platinum/cyclophosphamide chemotherapy, while GST pi was the predominant transferase. Mean GST activity and GST pi amount were decreased (P < 0.05) in malignant ovarian tumours after platinum/cyclophosphamide chemotherapy compared to untreated ovarian malignant tumours. No relation was found in untreated ovarian tumours between GST pi amount and response to platinum/cyclophosphamide chemotherapy. Thus, within the limitations of the current study no arguments were found for a role of GST in in vivo drug resistance of malignant ovarian tumours to platinum/cyclophosphamide chemotherapy.