Manon Verwijs-Janssen

The Baxα:Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia

Bcl‐2 over‐expression has been shown to inhibit apoptosis induced by a variety of stimuli, whereas a predominance of Baxα to Bcl‐2 accelerates apoptosis upon apoptotic stimuli. We sought to study the relevance of these apoptotic regulating gene products in leukaemia. In a panel of leukaemia and lymphoma cell lines (HL60, DoHH2, CEM C7, L1210 and S49), the Baxα‐to‐Bcl‐2 ratio as assessed by Western‐blot analysis correlated with sensitivity to dexamethasone treatment. In addition, in HAbaxα‐transfected CEM C7 clones, a similar correlation was found for dexamethasone and thapsigargin sensitivity. In bone‐marrow aspirates from patients with childhood acute lymphoblastic or myelocytic leukaemia (ALL, n = 48; AML, n = 8), the Bcl‐2 and Baxα levels were highly variable, but well within the range found in the Baxα transfectants and in the established cell lines. Bcl‐2 levels were lower in T‐ than in B‐lineage ALL, which could be ascribed to simultaneous inverse relation between Bcl‐2 and WBC. By contrast, Baxα:Bcl‐2 was independent of any presenting feature and was largely dependent on Baxα levels. Results suggest that Baxα:Bcl‐2, rather than Bcl‐2 alone is important for the survival of drug‐induced apoptosis in leukemic cell lines and ALL. Int. J. Cancer 71: 959‐965, 1997.

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells.

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase beta, comprising the 14 kDa DNA-binding domain of DNA polymerase beta but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5-1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G(1)-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase beta knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase beta-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta.

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polbetaDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polbetaDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polbetaDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H(2)O(2) treatment, polbetaDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H(2)O(2), these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polbetaDN induced increase in residual gammaH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual gammaH2AX foci, indicating an escape from gammaH2AX-mediated DSB repair. In addition, we show that in the polbetaDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.

Cell cycle phase dependent role of DNA polymerase β in DNA repair and survival after ionizing radiation

PURPOSE The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different phases of the cell cycle as determined by BrdU/flow cytometry. Cell kill and DNA repair were assessed by colony formation and alkaline comet assays, respectively. RESULTS We first demonstrated delayed repair of ionizing radiation induced DNA damage in confluent polymerase beta deficient cells. Cell synchronization experiments revealed a cell cycle phase dependence by demonstrating radiation hypersensitivity of polymerase beta-deficient cells in G1, but not in the S-phase. Complementing polymerase beta-deficient cells with polymerase beta reverted the hypersensitivity in G1. Ionizing radiation damage repair was found to be delayed in beta-deficient cells when irradiated in G1, but not in S. CONCLUSIONS The data show a differential role of DNA polymerase beta driven base excision and single strand break repair throughout the cell cycle after ionizing radiation damage.

Mutational analysis ofBax andBcl-2 in childhood acute lymphoblastic leukaemia

In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis‐regulating proteins Bax and Bcl‐2, but the molecular basis for this variation is unknown. Point‐mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl‐2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF (uORF) of bcl‐2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in bax and bcl‐2. DNA sequence analysis of the ORFs of bax and bcl‐2 and of the uORF of bcl‐2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl‐2 that (in)activate or deregulate Bax and Bcl‐2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease. Int. J. Cancer (Pred. Oncol.) 79:273–277, 1998.© 1998 Wiley‐Liss, Inc.

Fanconi anemia and homologous recombination gene variants are associated with functional DNA repair defects in vitro and poor outcome in patients with advanced head and neck squamous cell carcinoma

Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1–6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.