A. Binder

The role of G2-block abrogation, DNA double-strand break repair and apoptosis in the radiosensitization of melanoma and squamous cell carcinoma cell lines by pentoxifylline

Purpose : To examine the role of G2-block abrogation, DNA repair inhibition and apoptosis in the enhancement of radiotoxicity by pentoxifylline. Materials and methods : The influence of pentoxifylline on radiotoxicity was assessed by colony assay in TP53 wild-type Be11 and mutant MeWo melanoma, and in TP53 wild-type 4197 and mutant 4451 squamous cell carcinoma (SCC) cell lines. G2-block abrogation was assessed by flow cytometry. Induction of DNA damage and repair was measured over a dose range of 0-100Gy by constant field gel electrophoresis (CFGE). The Annexin-V binding assay was used to identify apoptotic cells. Results : Pentoxifylline, when combined with irradiation, significantly increased radiotoxicity in the TP53 mutant MeWo and 4451 cell lines by radiotoxicity enhancement factors of 3 and 14.5 respectively. No radiosensitization was seen in the TP53 wild-type Be11 and 4197 cells. When the drug was added after irradiation at the time of maximum G2-block expression, no radiosensitization was seen in any of the four cell lines. CFGE analyses showed that pentoxifylline effectively suppressed DNA double-strand break (DSB) repair in all four cell lines, as indicated by 20 h repair inhibition factors of 1.4-2.4. Pentoxifylline did not increase apoptosis in any of the four cell lines. Conclusion : These data suggest that radiosensitization by pentoxifylline is not a consequence of G2-block abrogation alone, but that inhibition of DSB repair plays a role in certain cell types.PURPOSE To examine the role of G2-block abrogation, DNA repair inhibition and apoptosis in the enhancement of radiotoxicity by pentoxifylline. MATERIALS AND METHODS The influence of pentoxifylline on radiotoxicity was assessed by colony assay in TP53 wild-type Bell and mutant MeWo melanoma, and in TP53 wild-type 4197 and mutant 4451 squamous cell carcinoma (SCC) cell lines. G2-block abrogation was assessed by flow cytometry. Induction of DNA damage and repair was measured over a dose range of 0-100 Gy by constant field gel electrophoresis (CFGE). The Annexin-V binding assay was used to identify apoptotic cells. RESULTS Pentoxifylline, when combined with irradiation, significantly increased radiotoxicity in the TP53 mutant MeWo and 4451 cell lines by radiotoxicity enhancement factors of 3 and 14.5 respectively. No radiosensitization was seen in the TP53 wild-type Be11 and 4197 cells. When the drug was added after irradiation at the time of maximum G2-block expression, no radiosensitization was seen in any of the four cell lines. CFGE analyses showed that pentoxifylline effectively suppressed DNA double-strand break (DSB) repair in all four cell lines, as indicated by 20 h repair inhibition factors of 1.4-2.4. Pentoxifylline did not increase apoptosis in any of the four cell lines. CONCLUSION These data suggest that radiosensitization by pentoxifylline is not a consequence of G2-block abrogation alone, but that inhibition of DSB repair plays a role in certain cell types.

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

Purpose : To determine the relationship between DNA damage and radiosensitivity at low doses (1-10Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. Materials and methods : Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60 Co γ-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). Results : The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r 2 =0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. Conclusion : It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.PURPOSE To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.

Chemosensitivity of prostatic tumour cell lines under conditions of G2 block abrogation.

Abstract Conventional chemotherapy has had very limited success in the control of hormone-refractory prostate cancer. Methylxanthine derivatives, such as pentoxifylline (PTX), are known to abrogate the G2 block and enhance the toxicity of ionising irradiation and chemotherapeutic agents. It is now also established that late addition of the cytotoxic drug after irradiation under conditions of G2 block abrogation sensitises human tumour cells for cytotoxins. Here we assess whether the chemosensitivity of prostate tumour cell lines can be enhanced by the application of a low dose of drug in conjunction with a G2 block abrogator. Prostate cell lines DU145, BM1604 and LNCaP were irradiated with 7 Gy 60Co γ-irradiation. A sub-toxic (2 mM) dose of pentoxifylline and a cytotoxic drug were added at maximum expression of the G2 cell cycle block and cell survival was determined by colony assay. Cisplatin, etoposide and vinblastine were tested at a toxic dose of 10% (TD10). In the TP53 mutant cell lines, DU145 and BM1604, dose enhancement factors (EFs) were found to be in the region of 4.20 for cisplatin, 3.70 for vinblastine, and 3.20 for etoposide. In the TP53 wild-type cell line, LNCaP, the enhancement factors were low and in the region of 1.20 for cisplatin, vinblastine and etoposide. It is clear, therefore, that toxicity enhancement factors (EFs) are greater in the TP53 mutant cell lines, DU145 and BM1604, than in the TP53 wild-type cell line, LNCaP. The results indicate that a significant enhancement of drug toxicity can be obtained if the cytotoxic drug is given under conditions of G2 block abrogation. The sensitisation of prostate cancer cells to cytotoxic drugs is particularly high in radiation-resistant TP53 mutant tumour cells. Drugs which abrogate G2 block have the potential to enhance the therapeutic index and therefore reduce the toxicity of chemotherapy drugs.

Abrogation of G2/M-Phase Block Enhances the Cytotoxicity of Daunorubicin, Melphalan and Cisplatin in TP53 Mutant Human Tumor Cells

Abstract Binder, A., Serafin, A. and Bohm, L. Abrogation of G2/M-Phase Block Enhances the Cytotoxicity of Daunorubicin, Melphalan and Cisplatin in TP53 Mutant Human Tumor Cells. Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40–60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G2-phase block, where the fraction of cells in G2 phase reaches a maximum after 18–20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G1- and G2-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G2 phase has reached a maximum shortens the normal recovery from G2-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3–2.8, 8.6–85 and 52–74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3–1.4, 2.3–3.0 and 1.2–2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G2-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.

Radiosensitization and DNA repair inhibition by pentoxifylline in NIH3T3 p53 transfectants

Purpose : To examine the role of p53 mutations in the modulation of DNA repair and radiotoxicity by pentoxifylline. Materials and methods : NIH3T3 murine cells transfected with mutant p53 constructs were examined for the influence of pentoxifylline on radiotoxicity to Co 60 γ-irradiation by colony assay. DNA repair (0-100 Gy) was measured by constant-field gel electrophoresis. Apoptosis was assessed by flow cytometry with the annexin-V-binding assay. Results : In the two p53 hot-spot mutant cell lines p53-S269R and p53- + 15, the SF 10 radiotoxicity enhancement factors induced by the pentoxifylline were 8.0 and 9.7, respectively. In the p53 deletion mutant p53- ΔA cell line, the radiotoxicity enhancement factor was 2.6. No radiosensitization was obtained in the untransfected p53 wild-type cell line U-Wt and in the transfected p53 double-wild-type p53-Wt cell line. When pentoxifylline was added after irradiation at the time of maximum G2 block expression, no radiosensitization was observed in any of the five cell lines. Constant-field gel electrophoresis analyses after 20 h of repair showed that pentoxifylline suppresses DNA double-strand break repair in all p53 mutant cell lines, as indicated by repair inhibition factors of 2.0-2.3. No repair suppression was found in the p53 wild-type cell lines. Conclusions : p53 mutations are a general requirement for radiosensitization by pentoxifylline and the level of radiosensitization depends upon the location of the p53 mutation.

Influence of pentoxifylline, A-802710, propentofylline and A-802715 (Hoechst) on the expression of cell cycle blocks and S-phase content after irradiation damage

The toxicity of the five methylxanthine derivatives, caffeine, pentoxifylline, A802710, propentofylline and A802715, was determined against the two human melanoma lines, Be11 and MeWo, and against the two human squamous cell carcinoma lines, 4197 and 4451, by vital dye staining assay. Pentoxifylline and A802710 emerge as the least toxic showing TD(50) (toxic dose of 50%) levels of 3.0-4.0 mM. Propentofylline and caffeine take an intermediate position. A802715 has a TD(50) of 0.9-1.1 mM and is the most toxic. Subtoxic concentrations (<TD50)added after irradiation at maximum expression of the G2/M block show that pentoxifylline and A802710 effectively abrogate the G2/M block, whereas A802715 and propentofylline prolong the G2/M block or remain ineffective depending on the p53 status of the cell line. In p53 wt cells BrdU incorporations show that the irradiation-induced suppression of S-phase entry is marginally enhanced by pentoxifylline but strongly enhanced by propentofylline and A802715. This effect was not seen in p53 mutant cells. Since propentofylline and A802715 prolong the G2/M block and effectively suppress BrdU incorporation these two drugs emerge as antagonists to pentoxifylline, caffeine and A802710. Common structural features of propentofylline and A802715 are a propyl substituent at the N7 position in contrast to pentoxifylline, caffeine and A802710 where the N7 substituent is a methyl group. The results document the effectiveness of four methylxanthines in influencing cell regulation and damage response in human tumor cells.

Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines

Phosphorylation of histone H3 at Ser‐10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co‐γ irradiation increases the number of cells binding anti‐histone H3‐P antibody 1–8‐fold in a p53‐independent manner. In the p53 mutant cell lines MeWo and 4451 H3‐P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post‐irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5–7 h post‐irradiation and then declines. These events are attributed to damage‐induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12–24 h post‐irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3–5‐fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3‐P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.

Differential effects of novel tumour-derived p53 mutations on the transformation of NIH-3T3 cells.

Abstract The p53 tumour suppressor gene is frequently mutated in human tumours and different tumour-derived mutations have varying effects on cells. The effect of a novel tumour-derived p53 mutation and two recently described mutations from South African breast cancer patients on the growth rate, colony formation, cell cycle arrest after irradiation and response to chemotherapeutic drugs was investigated. None of the p53 mutations had any significant effect on the inherent growth rate of the cells; however, contact inhibition of growth in two of the mutants was lost. These same two mutants formed colonies in soft agar, whereas the third mutant did not. All three of the mutants failed to show a G1 cell cycle arrest after exposure to 7 Gy of [60Co] radiation, albeit to different degrees. Cells expressing the p53 mutants were either more sensitive to cisplatin and melphalan or more resistant than the untransfected cells, depending on the mutation. However, there was no difference in response to daunorubicin treatment. These results demonstrate that different p53 mutations exert varying biological effects on normal cells, with some altering checkpoint activation more effectively than others. The data also suggest that the nature of the p53 mutation influences the sensitivity to cytostatic drugs.

Pentoxifylline Inhibits the Irradiation Induced G2/M Block and Alters DNA Synthesis in P53 Mutant and Repair Deficient Cells

The G1/S and G2/M transitions have been recognised as important checkpoints in the response of cells to sublethal damage. It is commonly thought that delays in the G1 or G2 progression which typically occurs 10-18 hrs postirradion are protective mechanisms which prevent propagation of defective DNA. However, this view may be simplistic and is at variance with the fact that rejoining of DNA strand breaks is complete within 3-4 hrs postirradiation.