Chris van Bree

Clonogenic assay of cells in vitro.

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose–survival curves is included.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41–42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

TR3 Orphan Receptor Is Expressed in Vascular Endothelial Cells and Mediates Cell Cycle Arrest

Objective—Endothelial cells play a pivotal role in vascular homeostasis. In this study, we investigated the function of the nerve growth factor–induced protein-B (NGFI-B) subfamily of nuclear receptors comprising the TR3 orphan receptor (TR3), mitogen-induced nuclear orphan receptor (MINOR), and nuclear orphan receptor of T cells (NOT) in endothelial cells. Methods and Results—The mRNA expression of TR3, MINOR, and NOT in atherosclerotic lesions was assessed in human vascular specimens. Each of these factors is expressed in smooth muscle cells, as described before, and in subsets of endothelial cells, implicating that they might affect endothelial cell function. Adenoviral overexpression of TR3 in cultured endothelial cells resulted in decreased [3H]thymidine incorporation, whereas a dominant-negative TR3 variant that inhibits the activity of endogenous TR3-like factors enhanced DNA synthesis. TR3 interfered with progression of the cell cycle by upregulating p27Kip1 and downregulating cyclin A, whereas expression levels of a number of other cell cycle–associated proteins remained unchanged. Conclusions—These findings demonstrate that TR3 is a modulator of endothelial cell proliferation and arrests endothelial cells in the G1 phase of the cell cycle by influencing cell cycle protein levels. We hypothesize involvement of TR3 in the maintenance of integrity of the vascular endothelium.

Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

PURPOSEnTo determine cross-resistance to anti-tumor treatments in 2,2difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells.nnnMETHODS AND MATERIALSnHuman lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes.nnnRESULTSnIn the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 microM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved.nnnCONCLUSIONSnIn the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation.

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH). It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.

Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins

Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21WAF1/CIP1 and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell‐cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis.

Hyperthermia and incorporation of halogenated pyrimidines: radiosensitization in cultured rodent and human tumor cells.

PURPOSEnTo investigate the possible benefit of hyperthermia (HT) in combination with radiosensitization by halogenated pyrimidines (HPs) in rodent as well as in human tumor cells.nnnMETHODS AND MATERIALSnExponentially growing rodent cells, radiosensitive R-1 and MOS cells and radioresistant RUC-II and V79 cells, and human SW1573 cells, were exposed to 0, 1, 2, and 4 microM of chloro- (CldUrd), bromo- (BrdUrd), or iodo-deoxyuridine (IdUrd) in the culture medium. Survival after irradiation with gamma-rays from a 137Cs source and/or hyperthermic treatment (HT, 60 min at 42 degrees C) was determined by clonogenic assay. Linear-quadratic analyses of the radiation survival curves were performed to assess sensitization in the dose range 1 to 3 Gy relevant to radiotherapy.nnnRESULTSnThe incorporation of HPs sensitized all cell lines to HT and resulted in radiosensitization dependent on the percentage of thymidine replacement. At equal levels of thymidine replacement, IdUrd was the most potent radiosensitizer. HT further increased radiation-induced lethality of cells that had incorporated HPs. Linear-quadratic analyses showed that HT further increased the linear parameter of the LQ formula while the quadratic parameter was not significantly changed.nnnCONCLUSIONnThe combination of HT and HPs act additively in increasing the radiosensitivity of rodent tumor cell lines with varying radiosensitivities as well as of a human tumor cell line. In particular, the ratio of the linear parameter to the quadratic parameter, relevant for fractionation effects in radiotherapy, was increased.

Colour junctions as predictors of radiosensitivity: X-irradiation combined with gemcitabine in a lung carcinoma cell line

PurposeTo determine whether measurement of colour junctions by fluorescent in situ hybridisation (FISH) can predict radiosensitisation of gemcitabine (2-2-difluorodeoxycytidine).MethodsHuman lung carcinoma cells (SW-1573) were irradiated with X-rays with or without incubation of 10xa0nM gemcitabine for 24xa0h. Cell survival was determined with clonogenic assay. Colour junctions were measured by whole chromosome FISH of chromosomes 2 and 18 and were scored according to the PAINT method.ResultsA clear radiosensitisation by gemcitabine was observed on cell survival. A significant decrease in the number of colour junctions was observed after gemcitabine treatment compared with radiation treatment alone. The correlation between colour junction induction and cell survival was high for both with and without gemcitabine, but the gemcitabine-sensitised curve did not coincide with the non-sensitised curve.ConclusionsGemcitabine-induced radiosensitisation is not predicted by induction of colour junctions in cultured SW-1573 cells. This reduces the clinical applicability of this predictive assay for radiotherapy in combination with gemcitabine.

Local hyperthermia enhances the effect of cis-diamminedichloro-platinum(II) on nonirradiated and preirradiated rat solid tumors

PURPOSEnWe have investigated differences in the efficacy of combined treatment with cis-diamminedichloroplatinum(II) (cDDP) and local hyperthermia (HT) in nonirradiated and preirradiated experimental tumors.nnnMETHODS AND MATERIALSnSurvival of R-1 rhabdomyosarcoma cells was assessed after treatment with various cDDP-concentrations at 37 degrees C and 42 degrees C in vitro. Rats bearing R-1 rhabdomyosarcomas of 190 mm3 (SE 15 mm3) were treated with cDDP (6 mg/kg i.p.), HT (1 h at 43 degrees C), or cDDP+HT (45 min interval) without preirradiation or at day 16 after the first dose of fractionated irradiation. Fractionated irradiation consisted of four daily doses of 5 Gy of x-rays each and tumor volumes had regrown to their original volume at the time of treatment. Experimental endpoint was tumor growth delay (TGD).nnnRESULTSnHyperthermia-enhanced cDDP cytotoxicity in vitro by a factor of about 5. Treatment with cDDP or HT alone resulted in a similar TGD in non- and preirradiated tumors (7.2 vs. 7.4 days and 1.1 vs. 0.9 days, respectively). In non- as well as in preirradiated tumors, HT given in combination with cDDP significantly enhanced the effect of cDDP, prolonging the TGD (11.1 days (p = 0.0001) and 16.2 days (p < 0.0001), respectively) corresponding to a TGD-enhancement of 1.54 and 2.19, respectively. The TGD after cDDP+HT in preirradiated tumors was significantly longer than in nonirradiated tumors (p = 0.0003).nnnCONCLUSIONSnIn this tumor model, HT enhanced the antitumor effect of cDDP. Previous radiation treatment did not reduce the HT-enhanced effect of cDDP. Combined cDDP and HT may be useful in the treatment of nonirradiated tumors as well as previously irradiated tumors.

Angiogenesis inhibitor DC101 delays growth of intracerebral glioblastoma but induces morbidity when combined with irradiation

The combination of irradiation with angiogenic inhibition is increasingly being investigated for treatment of glioblastoma multiforme (GBM). We investigated whether vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor DC101 affects morbidity and tumor growth in irradiated and non-irradiated intracerebral GBM-bearing mice, controlled with sham treatments. End-points were toxicity, morbidity and histology. Irradiation either or not combined, reduced tumor size strongly, whereas DC101 mono-treatment reduced tumor size by 64%. Irradiation delayed morbidity from 5.8 weeks in sham-treated mice to 10.3 weeks. Morbidity after combined treatment occurred after 5.9 weeks. Treatment with angiogenesis inhibitor DC101 delays tumor growth but it induces morbidity, by itself or combined with irradiation.