Gemcitabine uptake in glioblastoma multiforme: potential as a radiosensitizer.

Glioblastoma multiforme (GBM), the most frequent malignant brain tumor, has a poor prognosis, but is relatively sensitive to radiation. Both gemcitabine and its metabolite difluorodeoxyuridine (dFdU) are potent radiosensitizers. The aim of this phase 0 study was to investigate whether gemcitabine passes the blood-tumor barrier, and is phosphorylated in the tumor by deoxycytidine kinase (dCK) to gemcitabine nucleotides in order to enable radiosensitization, and whether it is deaminated by deoxycytidine deaminase (dCDA) to dFdU. Gemcitabine was administered at 500 or 1000 mg/m(2) just before surgery to 10 GBM patients, who were biopsied after 1-4 h. Plasma gemcitabine and dFdU levels varied between 0.9 and 9.2 microM and 24.9 and 72.6 microM, respectively. Tumor gemcitabine and dFdU levels varied from 60 to 3580 pmol/g tissue and from 29 to 72 nmol/g tissue, respectively. The gene expression of dCK (beta-actin ratio) varied between 0.44 and 2.56. The dCK and dCDA activities varied from 1.06 to 2.32 nmol/h/mg protein and from 1.51 to 5.50 nmol/h/mg protein, respectively. These enzyme levels were sufficient to enable gemcitabine phosphorylation, leading to 130-3083 pmol gemcitabine nucleotides/g tissue. These data demonstrate for the first time that gemcitabine passes the blood-tumor barrier in GBM patients. In tumor samples, both gemcitabine and dFdU concentrations are high enough to enable radiosensitization, which warrants clinical studies using gemcitabine in combination with radiation.

Molecular pathways involved in the synergistic interaction of the PKC beta inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells.

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKC beta inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription-PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3 beta and Akt phosphorylation, which was additionally reduced by drug combination (-58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients.

The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent.

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis. Simultaneous and sequential exposure (preexposure to 3-AP for 24h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC(25) concentration for 24h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR. Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR. In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.

Enhanced activity of deoxycytidine kinase after pulsed low dose rate and single dose gamma irradiation.

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.

Time course of enhanced activity of deoxycytidine kinase and thymidine kinase 1 and 2 in cultured human squamous lung carcinoma cells, SW-1573, induced by gamma-irradiation.

Single high dose rate irradiation of 4 Gy in SW-1573 cells, derived from non-small cell lung cancer, led to increased activities of deoxycytidine kinase (dCK) and thymidine kinase 1 and 2 (TK1 and 2). The activity of dCK increased by approximately 30% between 1 and 5 h after irradiation, after which the activity returned to the level of control cells by 8 h after irradiation. TK1 activity also increased by 30-50% between 1 and 6 h after irradiation. The decline to normal levels of dCK concurred with a further increase in the activity of TK1, 8 h after irradiation. TK2 activity was below control levels during the first 4 h after irradiation but rose 3-fold at 8 and 16 h after treatment. The activities of TK1 and TK2 had returned to approximate control levels 24 h after irradiation. The observation that mitochondrial TK2 activity increased to a very high level after irradiation may indicate that the activity of this enzyme is not only important for the damage to mitochondrial DNA, the increased activity may also be instrumental for repair of damage to nuclear DNA. We presume that the increase in activity of TK1 after irradiation is limited to cells in S-phase. Recruitment of cells into S-phase, to replace cells killed by irradiation, is probably too slow to offer an explanation for the enhanced activity of TK1 8 h after irradiation. The increase in activity of both dCK, and TK1 and 2 might be involved in an adaptive response of the cells to radiation by facilitation of DNA repair. The expression of protein kinase C (PKC) decreased during the first 5 h after irradiation. At 5 h after irradiation the level of expression had decreased by >50%. The decrease in PKC expression is concurrent with the increase in dCK activity. This suggests a role of PKC in the signal transduction from DNA damage to the increase in activity of enzymes instrumental in DNA repair.

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.

Quantitative real time PCR of deoxycytidine kinase mRNA by Light Cycler PCR; in relation to enzyme activity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleosides and various analogues such as gemcitabine (2',2'-difluorodeoxycytidine). We developed and optimized a sensitive real time Light Cycler (LC) PCR assay for dCK with SYBR green detection. The enzymatic activity measured in the same human xenografts of dCK correlated excellently with dCK mRNA expression levels measured by the LC. This assay can be used for evaluation of dCK expression in tumors.

Combinations of 5-fluorouracil with UCN-01 or staurosporine.

The action of 5-Fluorouracil (5-FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN-01, inhibitors of protein kinase C (PKC) on 5-FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5-FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5-FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2-fold higher than the sum of both drugs alone. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors seemed preferable.