Immunocytochemical detection of hENT1 and hCNT1 in normal tissues, lung cancer cell lines, and NSCLC patient samples.

Nucleoside transporters are essential for the cellular entry, efficacy, and cytotoxicity of several clinically important deoxynucleoside analogs (e.g., cytarabine and gemcitabine). We used immunohistochemistry to determine protein expression levels of the nucleoside transporters hENT1 and hCNT1 in NSCLC cell lines, NSCLC patient samples, and a variety of normal tissues. All 4 NSCLC cell lines expressed high to very high levels of both hENT1 and hCNT1. In NSCLC and normal tissues expression of hENT1 and hCNT1 ranged from completely negative to high. Immunohistochemistry might be a useful tool to predict response to deoxynucleoside analogs in malignancies treated with these drugs.

Potentiation of in vitro ara-C cytotoxicity by ribonucleotide reductase inhibitors, cyclin-dependent kinase modulators and the DNA repair inhibitor aphidicolin in paediatric acute myeloid leukaemia.

To modulate in vitro cytarabine (ara-C) resistance we combined ara-C with six potential resistance modifiers in 10 paediatric acute myeloid leukaemia (AML) patient samples (methyl thiazol tetrazolium assay). Drug interactions were determined by median drug effect analysis. Co-incubation of ara-C/aphidicolin showed strong synergism. The combinations of ara-C/cladribine and ara-C/gemcitabine were synergistic. Nearly additive and moderately synergistic interactions were observed between ara-C/flavopiridol and ara-C/UCN-01. The combination of ara-C/decitabine was antagonistic. In conclusion, favourable interactions were observed between ara-C and aphidicolin, cladribine, gemcitabine and also with flavopiridol and UCN-01, supporting the evaluation of these combinations in clinical trials with AML patients.

Immunocytochemical detection of deoxycytidine kinase in haematological malignancies and solid tumours.

BACKGROUND: Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important deoxynucleoside analogues used for the treatment of haematological and solid malignancies. AIM: To measure dCK expression in tumour cells from different origins. METHOD: A rabbit antihuman dCK antibody was used for the immunocytochemical detection of dCK expression in three leukaemic cell lines (HL60, U937, and CCRF-CEM) and 97 patient samples (paediatric acute myeloid leukaemia (AML) and lymphoid leukaemia (ALL), retinoblastoma, paediatric brain tumours, and adult non-small cell lung cancer (NSCLC)). RESULTS: CCRF-CEM, U937, and HL60 cells stained positively for dCK and the degree of expression correlated with dCK activity. dCK expression varied between tumour types and between individual patients within one tumour type. dCK was located predominantly in the cytoplasm. The staining intensity was scored as negative (0), low (1+), intermediate (2+), or high (3+). Expression of dCK was high in AML blasts. In contrast, brain tumour samples expressed low amounts of dCK. dCK staining ranged from low (1+) to high (3+) in ALL blasts, retinoblastoma, and NSCLC tissue samples. Staining was consistent (interobserver variability, 88%; kappa = 0.83) and specific. Western blotting detected the dCK protein appropriately at 30 kDa, without additional bands. CONCLUSIONS: Immunocytochemistry is an effective and reliable method for determining the expression of dCK in patient samples and requires little tumour material. This method enables large scale screening of dCK expression in tumour samples.

Immunocytochemical detection of deoxycytidine kinase in pediatric malignancies in relation to in vitro cytarabine sensitivity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of cytarabine (ara-C), a deoxycytidine analog active against acute leukemias. Resistance to ara-C has been linked to dCK deficiency. In this study we determined the expression of the dCK protein in pediatric malignancies, using immunocytochemistry and related the expression levels to in vitro ara-C sensitivity (measured with the MTT-assay). dCK expression was high in the AML and retinoblastoma samples, in the ALL samples dCK expression ranged from low to very high. The brain tumor samples expressed low levels of dCK. AML was significantly more sensitive in vitro to ara-C compared to ALL (p = 0.03). Retinoblastoma and brain tumor cells were extremely resistant in vitro, we were unable to detect more than 50% ara-C induced cell kill in the majority of samples. Samples were combined in groups according to dCK expression. Samples with low dCK expression were significantly more resistant to ara-C compared to samples with high dCK expression. In conclusion, dCK expression varies between individual samples and between different types of malignancies and may play a role in resistance to ara-C in particular tumor types.

Modulation of cytarabine induced cytotoxicity using novel deoxynucleoside analogs in the HL60 cell line.

In order to enhance the cytotoxicity of ara-C in the HL60 cell line the following deoxynucleoside analogs were used: cladribine, fludarabine and gemcitabine. HL60 cells were co-incubated with ara-C and each of the modulators at the ratios of their respective IC50s. Cytotoxicity was determined with the MTT-assay and drug interactions were evaluated with the combination index (CI) method (Calcusyn; Chou & Talalay). CI < 1, CI +/- 1 and > 1 indicate synergism, additive effect and antagonism, respectively. We observed moderate synergism between ara-C/cladribine and ara-C/gemcitabine, with CIs of 0.76 +/- 0.14 and 0.82 +/- 0.04, respectively. The interaction between ara-C/fludarabine resulted in moderate antagonism (CI = 1.29 +/- 0.11). In conclusion, in this in vitro study we showed that the cytotoxicity of ara-C can be succesfully modulated in the HL60 cell line by cladribine and gemcitabine.

In vitro cytotoxicity of aplidin and crossresistance with other cytotoxic drugs in childhood leukemic and normal bone marrow and blood samples: a rational basis for clinical development.

To determine the potential of aplidin as a cytotoxic agent in pediatric leukemia, we tested bone marrow (BM) and peripheral blood (PB) samples (n=72) of children with different types of leukemia and healthy children in the methyl-thiazol-tetrazolium assay. Also, we compared these results with other cytotoxic drugs. Aplidin was cytotoxic in vitro at nanomolar concentrations, in a dose-dependent fashion. L-carnitine, that is applied in clinical studies to prevent myotoxicity caused by aplidin, had no effect on aplidin cytotoxicity in vitro. Aplidin cytotoxicity in vitro was not different when initial and relapsed acute lymphoblastic leukemia (ALL) or initial ALL and initial acute myeloid leukemia were compared. However, normal BM (n=19) and PB (n=13) cells were more resistant to aplidin than leukemic cells (median two- to seven-fold, P=0.001 and median four- to 11-fold, P&<0.0001, respectively). In leukemia samples, no significant crossresistance between aplidin and other cytotoxic drugs was found, except for a trend for correlation with 2',2'-difluorodeoxycytidine (rho=0.71, P=0.02). In normal BM samples, significant crossresistance with the epipodophyllotoxins was found, which is not readily explained by the currently known mechanisms of action of aplidin. In conclusion, we show that aplidin has selective cytotoxicity in vitro towards childhood leukemia cells and generally lacks crossresistance with other known cytotoxic drugs, which warrants clinical studies.

Features of proliferation and in vitro drug resistance in central primitive neuro-ectodermal tumours.

The features of proliferation in brain tumours are related with clinical prognosis for several types of brain tumours, especially gliomas. For childhood central primitive neuro-ectodermal tumours (cPNET), including medulloblastoma, this relation has previously been unclear. The aim of this study is to investigate the relationship between proliferative features of cPNET and in vitro resistance for cytostatic drugs measured with the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide (MTT) assay. Tumour material was obtained from 23 surgical specimens of cPNET. The expression of the proliferation markers Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 was determined with immunohistochemistry, while S-phase and DNA ploidy were analysed by flowcytometric analysis cell scan (FACS). The in vitro resistance for 10 cytostatic drugs was determined with the MTT assay. Drug resistance levels were available in 19 (83%) of the 23 samples with a complete profile of 10 cytostatic drugs tested in 14 samples. An excellent correlation in drug resistance scores was found between pharmacologically related drugs. The Ki-67 staining in 20 samples varied from 10 to 60% and from 30 to 100% for PCNA. Cyclin D1 staining was negative in 11 out of 18 samples. The S-phase in 16 samples ranged from 2 to 16%. Increased staining of Ki-67 was related with actinomycin D sensitivity (r -.603; P=0.022), while cells with a higher S-phase percentage were more resistant to ifosfamide (r.952; P<0.0001). In vitro drug resistance testing of central primitive neuro-ectodermal tumours (PNET) is feasible with the MTT assay. Ifosfamide resistance was related with increased Ki-67 and S-phase percentage of the tumour cells, while increased Ki-67 was also related with actinomycin D sensitivity. These findings suggest a cell cycle dependent activity of cytostatic drugs in vitro.