Nicole Giocanti

Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis

Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1−/− 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1−/− 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1−/− 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1−/− 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content. [Mol Cancer Ther 2006;5(3):564–74]

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.

Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery

BackgroundThe cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair.ResultsPARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2.ConclusionsThe results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

Radiobiological Characterization of Two Therapeutic Proton Beams With Different Initial Energy Spectra Used at the Institut Curie Proton Therapy Center in Orsay

PURPOSE Treatment planning in proton therapy uses a generic value for the relative biological efficiency (RBE) of 1.1 throughout the spread-out Bragg peak (SOBP) generated. In this article, we report on the variation of the RBE with depth in the SOBP of the 76- and 201-MeV proton beams used for treatment at the Institut Curie Proton Therapy Center in Orsay. METHODS AND MATERIALS The RBE (relative to (137)Cs γ-rays) of the two modulated proton beams at three positions in the SOBP was determined in two human tumor cells using as endpoints clonogenic cell survival and the incidence of DNA double-strand breaks (DSBs) as measured by pulse-field gel electrophoresis without and with enzymatic treatment to reveal clustered lesions. RESULTS The RBE for induced cell killing by the 76-MeV beam increased with depth in the SOBP. However for the 201-MeV protons, it was close to that for (137)Cs γ-rays and did not vary significantly. The incidence of DSBs and clustered lesions was higher for protons than for (137)Cs γ-rays, but did not depend on the proton energy or the position in the SOBP. CONCLUSIONS Until now, little attention has been paid to the variation of RBE with depth in the SOBP as a function of the nominal energy of the primary proton beam and the molecular nature of the DNA damage. The RBE increase in the 76-MeV SOBP implies that the tumor tissues at the distal end receives a higher biologically equivalent dose than at the proximal end, despite a homogeneous physical dose. This is not the case for the 201-MeV energy beam. The precise determination of the effects of incident beam energy, modulation, and depth in tissues on the linear energy transfer-RBE relationship is essential for treatment planning.

Hyperfast, early cell response to ionizing radiation

Purpose : To determine whether the oscillatory changes of radiosensitivity which occur within fractions of a second to a few minutes following flash irradiation correlate with an altered incidence of apoptosis, DNA strand breaks or lipid-coupled signalling. Materials and methods : Human tumor cells (SQ-20B, LoVo) or Chinese hamster V79 fibroblasts were exposed to split-dose, pulse irradiation with 3.5 MeV electrons at high dose-rate (12 or 120Gys -1) and the effects assessed by clonogenic assays, analysis of DNA cleavage and microscopic observation. Results : The processes underlying oscillatory radiation response were saturable, but did not correlate with an increased incidence of DNA single- or double-strand breaks or apoptosis. N -acetylcysteine and inhibitors of lipid-derived signalling also failed to alter oscillatory response. However, this response did correlate with phenotypic alterations evoking mitotic or delayed cell death. Furthermore, high dose-rate irradiation provided a lower level of instability than protracted γ-ray irradiation. Conclusions : It is proposed that the early steps of DNA damage recognition and repair following priming radiation exposure bring about rapid, synchronous remodeling of chromatin, evoking enhanced chromosome damage upon re-irradiation.PURPOSE To determine whether the oscillatory changes of radio-sensitivity which occur within fractions of a second to a few minutes following flash irradiation correlate with an altered incidence of apoptosis, DNA strand breaks or lipid-coupled signalling. MATERIALS AND METHODS Human tumor cells (SQ-20B, LoVo) or Chinese hamster V79 fibroblasts were exposed to split-dose, pulse irradiation with 3.5 MeV electrons at high dose-rate (12 or 120 Gy x s(-1)) and the effects assessed by clonogenic assays, analysis of DNA cleavage and microscopic observation. RESULTS The processes underlying oscillatory radiation response were saturable, but did not correlate with an increased incidence of DNA single- or double-strand breaks or apoptosis. N-acetylcysteine and inhibitors of lipid-derived signalling also failed to alter oscillatory response. However, this response did correlate with phenotypic alterations evoking mitotic or delayed cell death. Furthermore, high dose-rate irradiation provided a lower level of instability than protracted gamma-ray irradiation. CONCLUSIONS It is proposed that the early steps of DNA damage recognition and repair following priming radiation exposure bring about rapid, synchronous remodeling of chromatin, evoking enhanced chromosome damage upon re-irradiation.

Radiation-induced arrest of cells in G2 phase elicits hypersensitivity to DNA double-strand break inducers and an altered pattern of DNA cleavage upon re-irradiation

Purpose : To determine how radiation-induced arrest in G2 affects the response of mammalian cells to a challenging dose of radiation or to antitumour drugs producing DNA double-strand breaks. Materials and methods : V79 fibroblast survival to 5 Gy γ-rays followed at intervals by 3 Gy irradiation or by contact with an equitoxic dose of neocarzinostatin or etoposide, was measured by clonogenic assays. The pattern of radiation-induced DNA double-strand breaks was determined by filter elution and CFGE (continuous field gel electrophoresis) or PFGE (pulsed-field gel electrophoresis) in G2-arrested cells as well as in nonpre-irradiated asynchronous or synchronized cells. The cell-cycle phase specificity of drug susceptibility was determined in synchronized HeLa cells. Results : Cell kill by radiation-drug combined treatment varied markedly with the time elapsed after priming irradiation. Pre-irradiated, G2-arrested V79 fibroblasts demonstrated excess double-stranded DNA cleavage upon re-irradiation and hyper-sensitivity to drugs and radiation, although maximum resistance to both neocarzinostatin and etoposide in synchronized HeLa cells was in G2. This effect occurred in the megabase range only, with a peak around 4 Mbp; no change in the electrophoretic migration profile of DNA was observed below 1 Mbp. Moreover, the DNA migration profile and the yield of DNA cleavage in G2-arrested cells were close to those expected from S-phase cells. Conclusion : The available data suggest that mechanisms operating within the radiation-induced G2 block promote susceptibility to DNA double-strand break inducers at this stage. It is also proposed that the conformation of DNA in cells accumulated in G2 following irradiation bears resemblance to that for cells in S phase, due either to active repair mechanisms or to inhibition of chromosome disentanglement at the S-G2 transition.PURPOSE To determine how radiation-induced arrest in G2 affects the response of mammalian cells to a challenging dose of radiation or to antitumour drugs producing DNA double-strand breaks. MATERIALS AND METHODS V79 fibroblast survival to 5 Gy gamma-rays followed at intervals by 3 Gy irradiation or by contact with an equitoxic dose of neocarzinostatin or etoposide, was measured by clonogenic assays. The pattern of radiation-induced DNA double-strand breaks was determined by filter elution and CFGE (continuous field gel electrophoresis) or PFGE (pulsed-field gel electrophoresis) in G2-arrested cells as well as in nonpre-irradiated asynchronous or synchronized cells. The cell-cycle phase specificity of drug susceptibility was determined in synchronized HeLa cells. RESULTS Cell kill by radiation-drug combined treatment varied markedly with the time elapsed after priming irradiation. Pre-irradiated, G2-arrested V79 fibroblasts demonstrated excess double-stranded DNA cleavage upon re-irradiation and hypersensitivity to drugs and radiation, although maximum resistance to both neocarzinostatin and etoposide in synchronized HeLa cells was in G2. This effect occurred in the megabase range only, with a peak around 4 Mbp; no change in the electrophoretic migration profile of DNA was observed below 1 Mbp. Moreover, the DNA migration profile and the yield of DNA cleavage in G2-arrested cells were close to those expected from S-phase cells. CONCLUSION The available data suggest that mechanisms operating within the radiation-induced G2 block promote susceptibility to DNA double-strand break inducers at this stage. It is also proposed that the conformation of DNA in cells accumulated in G2 following irradiation bears resemblance to that for cells in S phase, due either to active repair mechanisms or to inhibition of chromosome disentanglement at the S-G2 transition.

Growth inhibition by STI571 in combination with radiation in human chronic myelogenous leukemia K562 cells

Altered radiation responses by STI571 (Imatinib, Glivec), a specific inhibitor of the tyrosine kinase activity of Bcr-Abl, was assessed in K562 chronic myelogenous leukemia cells using growth inhibition and colony formation assays. Flow cytometry, Western blotting, and microscope observation were used to determine cell cycle redistribution, erythroid differentiation, apoptosis, necrosis, senescence, and expression and phosphorylation of effectors downstream from Bcr-Abl as endpoints. STI571 (≥24-h contact) retarded the growth of K562 cells and elicited reduction in the G2-phase content due to an efficient arrest in early S phase rather than to the disruption of the G2 checkpoint as confirmed by analysis of Lyn and CDK1 phosphorylation. STI571 brought about the inhibitory dephosphorylation of Bcr-Abl and STAT5, but the expression of DNA-PKcs and Rad51 was unaffected and the interaction between radiation and STI571 was strictly additive with regard to induction of apoptosis. Overall STI571 interacted cooperatively with radiation to retard the growth of K562 cells but did not affect intrinsic radiosensitivity. However, STI571 and radiation acted antagonistically with each other with regard to induction of senescence and erythroid differentiation. [Mol Cancer Ther 2008;7(2):398–406]

Antagonistic interaction between bicalutamide™ (Casodex®) and radiation in androgen-positive prostate cancer LNCaP cells

Bicalutamide™ (Casodex®) reportedly improves high‐risk prostate cancer survival as an adjuvant treatment following radiotherapy. However, biological data for the interaction between bicalutamide and ionizing radiation in concomitant association are lacking.

A role for PKCζ in potentiation of the topoisomerase II activity and etoposide cytotoxicity by wortmannin

Enhanced cytotoxicity of etoposide by wortmannin, an inhibitor of enzymes holding a phosphatidylinositol 3-kinase domain, was investigated in eight cell lines proficient or deficient for DNA double-strand break repair. Wortmannin stimulated the decatenating activity of topoisomerase II, promoted etoposide-induced accumulation of DNA double-strand breaks, shifted the specificity for cell killing by etoposide from the S to G1 phase of the cell cycle, and potentiated the cytotoxicity of etoposide through two mechanisms. (a) Sensitization to high, micromolar amounts of etoposide required integrity of the nonhomologous end-joining repair pathway. (b) Wortmannin dramatically increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. It is shown that this process correlates depression of phosphatidylinositol 3-kinase–dependent phosphorylation of the atypical, ζ isoform of protein kinase C (PKCζ). Stable expression of a dominant-negative, kinase-dead mutant of PKCζ in a tumor cell line reproduced the hypersensitivity pattern induced by wortmannin. The results are consistent with up-regulation of the topoisomerase II activity in relation to inactivation of PKCζ and indicate that PKCζ may be a useful target to improve the efficiency of topoisomerase II poisons at low concentration.

In Vitro Evaluation of a Novel 2,6,9‐Trisubstituted Purine Acting As a Cyclin‐Dependent Kinase Inhibitor

NICOLE GIOCANTI,a RAMIN SADRI,a MICHEL LEGRAVEREND,b ODILE LUDWIG,b EMILE BISAGNI,bSOPHIE LECLERC,c LAURENT MEIJER,c AND VINCENT FAVAUDONa.d aU 350 INSERM and bUMR 176 CNRS, Institut Curie, Centre Universitaire, 91405 Orsay, France cUPR 9042 CNRS, Station Biologique, 29682 Roscoff, France The frequent deregulation of cell cycle progression in cancer1 has prompted an active search for kinase inhibitors with high affinity and specificity for cyclin-dependent kinases (Cdks). Three major classes of Cdk-targeting drugs have been identified to date, including butyrolactone I,2 polyhydroxylated flavones such as flavopiridol,3 and substituted purines.4 The first substituted purine derivative acting as a selective Cdk inhibitor, olomoucine, has been identified from screening against Cdk1/cyclin B complex.5 Olomoucine competitively inhibits Cdk1, Cdk2, Cdk5, and, to a lesser extent, Erk1.5 Recent results have pointed to unexpected pharmacologic properties of 2,6,9trisubstituted purines derived from the olomoucine lead structure.6,7 To investigate the question in more detail, we developed a program for synthesis and evaluation of new compounds in this series. Twenty-seven derivatives were synthesized and assayed for specific inhibition of Cdk1/cyclin B from starfish oocytes and human recombinant Cdk5/p35 complex. In agreement with earlier results,5 data showed that a strong correlation exists between inhibitory efficiencies against Cdk1 and Cdk5. In contrast, all compounds were only marginally active against Erk1 and Erk2 kinases. One compound in the series, ML-1437, proved much more active than olomoucine against purified Cdk1/cyclin B, Cdk5/p35, and Cdk2/cyclin E. It also showed pronounced cytotoxicity against human cervix carcinoma HeLa cells in vitro, even on short exposure. Growing IMR-90 (human normal fibroblasts), LoVo (human colon adenocarcinoma), and SQ-20B (human head and neck squamous carcinoma) cells gave similar results, but drug resistance increased rapidly as cells (SQ-20B and IMR-90) reached confluence. These results suggest that the affinity for Cdks and the cytotoxic potential of the drugs are interrelated (FIG. 1, TABLE 1). With the exception of pronounced lengthening of S phase transit during early-S in synchronized HeLa cells, ML-1437 at subtoxic concentration proved unable to produce reversible arrest of the cell cycle progression. When observed, arrest in the G1 and G2 phases of the cell cycle correlated with induced cell death, and chronic exposure to lethal doses of the drug resulted in massive micronucleation in relation to mitotic cell death, with no evidence of endoreduplication (polyploidization) or ap-