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Programmed Cell Death (PCD) is a highly regulated but not yet fully understood process by which selective gene expression leads to cell demise. The term apoptosis refers to the typical morphological changes observed with PCD. In the present study, we investigated the potential role of apoptosis as a mechanism by which anti-estrogens mediate tumor regression. Estrogen receptor-positive MCF-7 human mammary adenocarcinoma cells were treated with three different estrogen antagonists: RU 58668, ICI 182,780, and 4OH-Tam, in order of increasing potency. At selected time intervals, for up to seven days of culture, the degree of apoptosis induced by the anti-estrogens was assessed. Apoptotic cells were scored on the basis of specific morphological criteria disernable by fluorescence microscopy. It was hypothesised that the more potent the anti-estrogen, the greater the number of apoptotic cells we would observe. However, only minimal apoptosis was noted even at the highest concentration of anti-estrogen assayed (10-5 M). This is in contrast to some, but not all, previous reports. Possible confounding factors are discussed and include the presence of exogenous estrogen in the culture media, as well as a genetic drift in the clonal lineage of MCF-7 cells such that a resistance to induced apoptosis had been acquired.

The high porosity and versatile composition of the benchmarked mesoporous metal (Fe, Al, Cr) trimesate metal–organic frameworks (MIL-100(Fe, Al, Cr)) make them very promising solids in different strategic industrial and societal domains (separation, catalysis, biomedicine, etc.). In particular, MIL-100(Fe) nanoparticles (NPs) have been recently revealed to be one of the most promising and innovative next generation tools enabling multidrug delivery to overcome cancer resistance. Here, we analyzed the in vitro toxicity of the potential drug nanocarrier MIL-100(Fe) NPs and the effect of the constitutive cation by comparing its cytotoxicity with that one of its Cr and Al analogue NPs. Lung (A549 and Calu-3) and hepatic (HepG2 and Hep3B) cell lines were selected considering pulmonary, ingestion or intravenous exposure modes. First, the complete physicochemical characterization (structural, chemical and colloidal stability) of the MIL-100(Fe, Al, Cr) NPs was performed in the cell culture media. Then, their cytotoxicity was evaluated in the four selected cell lines using a combination of methods from cell impedance, cell survival/death and ROS generation to DNA damage for measuring genotoxicity. Thus, MIL-100(Fe, Al, Cr) NPs did not induce in vitro cell toxicity, even at high doses in the p53 wild type cell lines (A549 and calu-3 (lung) and HepG2 (liver)). The only toxic effect of MIL100-Fe was observed in the hepatocarcinoma cell line Hep3B, which is stress sensitive because it does not express TP53, the guardian of the genome.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

Having in mind the huge potential of ND for numerous medical applications including, among others, drug delivery, medical imaging, molecular targeting, and implants, it is crucial to study if they present a potential risk for human health. This chapter describes what is currently known regarding the in vitro cytotoxicity and genotoxicity of ND on human differentiated cells and on stem cells, and on the interaction of ND with biomolecules and especially with blood proteins. Although data are still fragmented, a point is also made regarding in vivo animal models, biokinetics, and bioaccumulation of NDs, and their potential hazardous effects on retention compartments/organs. Overall, the absence of obvious acute adverse effects of ND on in vitro and on experimental animals, indeed need further confirmation, but published data indicate that NDs are very promising for new nanotechnologies and methods for medicine.

Aberrant telomere structure is characteristic of resistant chronic lymphocytic leukaemia cells

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-κB (RELB, BCL3), Wnt, TGFβ, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.

International audience; Hydrogenated nanodiamonds (H-NDs) exhibit a negative electron affinity that confers a high reactivity with oxygen species and a positive charge in aqueous solutions. It allows electron emission from H-NDs following irradiation by photons and in consequence may enhance the effects of radiation on cancer cells. By using three human radioresistant cancer cell lines, we showed a potentialization of cytotoxicity after a co-exposure to H-NDs and irradiation; an event occurring through the induction of DNA damage and reactive oxygen species. This occurred together with a decrease in cell impedance, the activation of G(1)/S, an unlocking of G(2) cell cycle check-points and early low cell death rate. At later stage of exposure, persistent increases in heterochromatinization, large gamma-H2AX foci and p-galactosidase activity were detected providing evidence of cells' entrance into senescence. Similar potentialization was observed with neocarzinostatin (NCS), a radiomimetic drug. This original finding underlines a wide clinical potential of H-NDs to intensify radiation effects on radio-resistant cancer cells.

Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome.

B-chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course which has long remained a stumbling block for clinicians. This variability appears to arise from complex molecular alterations identified in malignant cells from patient subsets. Recent studies have focused in particular on identifying new molecular markers to help predict the most effective and adapted treatments. In addition to the mutation status of immunoglobulin variable heavy-chain region (IgVH) genes, which is a well-established predictive factor in B-CLL, these new markers include defects of cell factors involved in the maintenance of genome stability, such as telomere function, DNA repair, ATM and p53. Other predictive factors, such as tyrosine kinase Zap-70 and soluble factors found in patient sera, may be associated with B-cell receptor signal transduction. Interestingly, an alteration of these factors fits closely, though not strikingly, with the absence of somatic mutations in IgVH genes, suggesting that the latter may be due either to epigenetic events leading to an unstable genome or to an inherited defect in the immune response of malignant B-cells. Recent lessons from Zap-70 expression/phosphorylation suggest that some of these markers may reflect the defective pathways in B-CLL cells rather than being markers of cell malignancy per se. Furthermore, specific subsets of markers are found in patient cells resistant to treatment. Current studies on gene expression profiling and proteomic analyses should soon lead to a better understanding of how these pathways are affected, especially in multi-drug resistant B-CLL.

Human cancer is characterised by complex molecular aberrations which result in a wide variety of clinical manifestations. B-cell chronic lymphocytic leukaemia (B-CLL) is particularly diverse, both in terms of molecular changes and clinical course, and consequently our understanding of the pathology of this disease is generally poor. Furthermore, the heterogeneity of this tumour type coupled with the absence of an obvious genetic "hallmark", such as gain of oncogene function or loss of suppressor-gene function, has led many investigators to question whether B-CLL is a single disease entity. In most cases, B-CLL does not show specific reciprocal chromosomal translocations as found in other haemopoietic malignant diseases. The genomic instability of B-CLL results in numerous different types of chromosomal losses and gains, giving rise to unsettled karyotypes among individuals with this disease. Nevertheless, genetic data imply that B-CLL is a single disease characterised by a common gene-expression profile and by the existence of specific subtypes that may have clinical correlates in patients.

Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3′5′ cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-α (SDF-1α). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.

The EP4 receptor conveys growth-inhibitory effects in mature and immature B cells via NF-κB. Herein, the EP4 receptor was evaluated as a potential therapeutic target for leukemia and lymphoma, whose survival depends on the constitutive activity of NF-κB. Utilizing a pharmacological approach, we proved that the EP4 receptor induces caspase-mediated apoptosis in malignantly transformed B cells, with the most prominent effect being on Burkitt׳s lymphoma cells. Since the increased activation of NF-κB underlies multi-drug resistance phenomena, we modulated this signaling pathway via EP4 receptor triggering. Pge1-OH, a specific EP4 receptor agonist, led to decreased NF-κB activity and a consequent decrease in levels of the antiapoptotic gene Bcl-xL in Ramos cells, resulting in an elevated sensitivity of cells towards bortezomib- and doxorubicin-induced chemotherapeutic effects. Our study identifies the as yet unrecognized potential of EP4 receptor agonists as chemo-sensitizing agents in B-cell lymphoma. The specific downregulation of NF-κB-dependent pathways in B-cell malignancies opens new possibilities for treatment and current therapy optimization using specific EP4 receptor agonists.

We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' - 5' DNA helicase identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modifications in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process. Oncogene (2000).

Ubiquitin-proteasome-dependent protein processing appears to be an essential component in the control of radiation-induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radio-resistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF-κB or the cytoplasmic level of IκBα remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome-ubiquitin-dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.

Fine-needle samples of 75 non-Hodgkin's lymphomas were investigated for apoptosis immediately and after 24 h of culture after in vitro irradiation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells were simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub-G1 peak cells. The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed immediately (9% by morphology vs. 10% by flow) or after 24 h of culture (40% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by flow cytometry (64% by morphology vs. 55% by flow after 2 Gy irradiation, and 71% vs. 58% after 10 Gy). The results of the two methods were correlated, although large differences were seen between the techniques in individual tumors. In our system, flow cytometric sub-G1 peak analysis appears to underestimate apoptosis. Of these two methods, we find the Hoechst morphology method to be more reliable for quantitation of apoptosis utilizing fresh fine-needle sample material, in that discrimination of apoptotic cells from debris is easier and that both early and late apoptotic cells are detectable.

Apoptosis can be triggered by cytotoxic agents and radiation currently used in cancer treatment. However, the apoptotic response appears to vary between cell types (normal or transformed) and between types of malignancy. Thus, irradiation induces apoptosis in normal human lymphocytes but not in lymphocytes derived from a subset of chronic lymphocytic leukaemia (CLL). Moreover, in this subset, spontaneous apoptosis is inhibited by irradiation. Why irradiation does not allow the initiation of the apoptotic death pathway could be explained, at least in part, and in agreement with recent findings on experimental models, by the activation of the transcriptional factor NF-kappaB, which is able to inhibit apoptotic cell response. Low doses (at which no effect is observed with normal human lymphocytes) of the highly specific proteasome inhibitor lactacystin are sufficient to trigger apoptosis in these malignant cells. Proteasome inhibition by lactacystin prevents the nuclear translocation of both p50 and p65 NF-kappaB subunits and sensitizes these cells to apoptosis by tumour necrosis factor (TNF)-alpha treatment. As this subset of CLL is totally resistant to any treatment, proteasome inhibition by lactacystin provides a new therapeutic approach to be explored, considering the sensitivity of malignant CLL-derived lymphocytes to be quite different from that of normal human lymphocytes. Images Figure 1 Figure 3

Ionizing radiations have been reported as an in vitro apoptosis initiating stimulus in human lymphocytes. As the cytotoxicity of ionizing radiations and chemotherapeutic agents appears to be dependent on the efficacy of cell death induction, the manipulation of apoptosis initiation might be used as a means to supress some pathological process. In the present study the in vivo induction of gamma-ray mediated programmed cell death in humans is reported. The in vivo induction of apoptosis in peripheral blood lymphocytes (PBL) by ionizing radiations was investigated in 33 patients after each of two sessions (2 Gy and 4 Gy) of fractionated total body irradiation (FTBI) as part of their conditioning regimen before bone marrow transplantation. PBL committed to apoptosis were scored before irradiation (S1), 4 h (S2) and 24 h after 2 Gy (S3, 14-17 h after the second 2 Gy fraction). Nuclear morphology and chromatin-DNA were analysed by fluorescence microscopy immediately after blood sample withdrawal (I) and after 24 h in cell culture medium (II). When scored immediately after withdrawal, no circulating PBL with the apoptotic nuclear morphology were observed in S1 and S2 blood samples whereas S3 disclosed 21.9 +/- 11.7% of circulating lymphocytes with an apoptotic nuclear morphology. After 24 h in culture, S1 samples (before irradiation) generally contained less than 20% of apoptotic lymphocytes. A higher percentage of apoptotic cells was noted in some cases in relation with recent chemotherapy and possibly with pathology. After 24 h in culture, S2 and S3 samples contained 51.7 +/- 17.9% and 60.4 +/- 16.4% of apoptotic lymphocytes, respectively. These results confirm that ionizing radiations induce apoptosis in vivo in human lymphocytes and that the commitment to apoptosis can be determined after low doses (2 Gy) of therapeutic whole body irradiation. The results suggest that susceptibility to apoptosis induction by ionizing radiations could be related to previous therapy by cytotoxic drugs and possibly to the type of haematological malignancy.

Jozo Delic1, Jean-Brice Marteau1, Karim Maloum2, Florence Nguyen-Khac2, Frederic Davi2, Zahia Azgui2, Veronique Leblond3, Jacques-Louis Binet2, Sylvie Chevillard1 and Helene Merle-Beral 1Laboratoire de Cancerologie Experimentale, Institut de Radiobiologie Cellulaire et Moleculaire, Commissariat a l’Energie Atomique et aux Energies Renouvelables (CEA), 2Services d’Hematologie Biologique, Groupe Hospitalier Pitie-Salpetriere, 3Clinique, Groupe Hospitalier Pitie-Salpetrier, France

In somatic cells, eroded telomeres can induce DNA double-strand break signaling, leading to a form of replicative senescence or apoptosis, both of which are barriers to tumorigenesis. However, cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis, enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction, and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3′ single-stranded overhangs and an increased number of telomere-induced foci containing γH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs, 2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably, these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions, or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together, our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction.

Background The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes. Methods Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test. Results Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells. Conclusion The molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in males.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K−) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

In this study, we explored the telomeric changes that occur in B-chronic lymphocytic leukemia (B-CLL), in which telomere length has recently been demonstrated to be a powerful prognostic marker. We carried out a transcriptomic analysis of telomerase components (hTERT and DYSKERIN), shelterin proteins (TRF1, TRF2, hRAP1, TIN2, POT1, and TPP1), and a set of multifunctional proteins involved in telomere maintenance (hEST1A, MRE11, RAD50, Ku80, and RPA1) in peripheral B cells from 42 B-CLL patients and 20 healthy donors. We found that, in B-CLL cells, the expressions of hTERT, DYSKERIN, TRF1, hRAP1, POT1, hEST1A, MRE11, RAD50, and KU80 were more than 2-fold reduced (P < .001), contrasting with the higher expression of TPP1 and RPA1 (P < .001). This differential expression pattern suggests that both telomerase down-regulation and changes in telomeric proteins composition are involved in the pathogenesis of B-CLL.

Nonhomologous end-joining (NHEJ) DNA factors maintain genomic stability through their DNA double-strand break (DSB) repair and telomere-associated activities. Unrepaired or misrepaired DSBs can lead to apoptotic death or chromosomal damage. The B cells of some B-chronic lymphocytic leukemia (B-CLL) patients are resistant to radiation-induced apoptosis in vitro. We show here that the novel DNA-dependent protein kinase (DNA-PK) inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), and the phosphatidylinositol 3 (PI-3) kinase inhibitor, wortmannin, restored sensitivity to DNA damage-induced apoptosis of otherwise resistant cells. These resistant malignant B cells also escaped DSB-induced apoptosis following exposure to etoposide or neocarzinostatin. We found that at 15 minutes after irradiation, the levels of NHEJ (as measured by an in vitro DSB end-ligation assay) and DNA-PK catalytic subunit (DNA-PKcs) activity were, respectively, 2-fold and 4-fold higher in radio-resistant than in radio-sensitive B-CLL cells or Epstein-Barr virus (EBV)-transformed B cells. Ku70/Ku80 heterodimer DNA end-binding activity was also 2- to 3-fold higher in the resistant B-CLL cell subset compared with the sensitive B-CLL cell subset. Our results provide the first evidence that overactivating the NHEJ DNA repair pathway impairs DNA damage-induced apoptosis in malignant B cells and that this may contribute to their resistance to current chemotherapy. (Blood. 2005;105:4776-4783)

B-cell chronic lymphoid leukemia (BCLL) is a highly heterogeneous human malignancy, presumably reflecting specific molecular alterations in gene expression and protein activity that are thought to underlie the variable disease outcome. Most B-CLL cell samples undergo apoptotic death in response to DNA damage. However, a clinically distinct aggressive subset of B-CLL is completely resistant in vitro to irradiation-induced apoptosis. We therefore addressed 2 series of microarray analyses on 4 sensitive and 3 resistant B-CLL cell samples and compared their gene expression patterns before and after apoptotic stimuli. Data analysis pointed out 16 genes whose expression varied at least 2-fold specifically in resistant cells. We validated these selected genes by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) on 7 microarray samples and confirmed their altered expression level on 15 additional B-CLL cell samples not included in the microarray analysis. In this manner, in 11 sensitive and 11 resistant B-CLL cell samples tested, 13 genes were found to be specific for all resistant samples: nuclear orphan receptor TR3, major histocompatibility complex (MHC) class II glycoprotein HLA-DQA1, mtmr6, c-myc, c-rel, c-IAP1, mat2A, and fmod were up-regulated, whereas MIP1a/GOS19-1 homolog, stat1, blk, hsp27, and ech1 were down-regulated. In some cases, the expression profile may be dependent on the status of p53. Some of these genes encode general apoptotic factors but also exhibit lymphoid cell specificities that could potentially be linked to the development of lymphoid malignancies (MIP1alpha, blk, TR3, mtmr6). Taken together, our data define new molecular markers specific to resistant B-CLL subsets that might be of clinical relevance.

In order to identify CLL patients for whom irradiation could be beneficial, we investigated the relationship between in vitro radio-induced apoptosis of leukemic cells and response to low-dose splenic or lymph node radiotherapy. Fourteen patients were included in the in vitro study. Leukemic cells were analyzed by Hoechst staining immediately after collection or 24 h of culture following in vitro irradiation of 0-10 Gy. The tumor response rate was 47% (one CR and six PR), with a mean duration of response of 3 months (range: 1-4). A high correlation between tumor response and in vitro tests was observed (p

The ubiquitin system regulates diverse biological processes such as DNA replication and repair, biogenesis of ribosome, peroxisome and nucleosome, cell cycle, stress response and signal transduction pathways. Thus, the reported role of the ubiquitin system in apoptotic death control as well the alteration of its control in carcinogenesis should come as no surprise. Indeed, we and other groups have reported that the ubiquitin system is involved in apoptotic cell death of normal human lymphocytes and that this control is altered in B lymphocytes derived from chronic lymphocytic leukemia patients (B-CLL), rendering these malignant cells hypersensitive to specific inhibition of protein degradation/processing through proteasomal function. This approach recently allowed us to demonstrate that the stability of the tumor suppressor and pro-apoptotic protein p53 is differentially regulated in B-CLL versus normal lymphocytes and that this difference might at least partly explain the impaired response of B-CLL lymphocytes to apoptotic death activation. These results strongly suggest an imbalance in p53 regulation in B-CLL cells that leads to a variable response to DNA damage and constitutively expressed chromosomal instability. The question we and others would like to address is whether this alteration, or more likely a subset of alterations of the ubiquitin-proteasome pathway, is specific to B-CLL malignancy or if it is a hallmark of cancer cells in general. In either case, a better understanding of the ubiquitin-dependent control of apoptosis should pave the way towards a methodological approach for in vitro development of discriminating treatments which may be of potential usefulness in clinical trials of B-CLL.

The ubiquitin-proteasome-dependent proteolytic system has been reported to regulate apoptotic cell death in many experimental cell models. We recently found that B-CLL (chronic lymphocytic leukemia) lymphocytes are hypersensitive to apoptotic death activation through specific inhibition of proteasome function by lactacystin. Lactacystin efficiently activates apoptotic death process in B-CLL lymphocytes at doses at which no apoptotic effect can be observed in normal human lymphocytes in which 10-fold higher doses of lactacystin are required to weakly induce apoptosis. This hypersensitivity of B-cell CLL may be a result of an altered ubiquitin pathway and proteasomal proteolysis in these malignant cells, and this alteration could be specific for this malignancy. Together with other published works, these results suggest that lactacystin, though not per se a discriminatory inhibitor of the ubiquitinated protein processing/degradation, can nonetheless be discriminatory in the apoptotic cell response between B-CLL and normal lymphocytes: the property that promises efficacy in clinical trials of B-cell CLL. This hypothesis is documented by the fact that lymphocytes from patients in complete remission become resistant to lactacystin-induced apoptosis as normal lymphocytes do.

The ubiquitination of nuclear proteins activated in human lymphocytes undergoing radiation-induced apoptosis and the subsequent downstream proteasomal protein processing, shown to be involved in apoptotic death control, may be dependent on an amino-terminal sequence identity of ubiquitin target proteins, the “N-end rule” pathway. Here we report that this selective pathway controls radiation-induced apoptosis and that it is involved in the initiation of this type of cell death. Dipeptide competitors of protein ubiquitination/processing dependent solely on the basic amino-terminal residues (type I) efficiently inhibited the radiation-induced apoptotic death phenotype, indicating that only the substrates of ubiquitination with basic NH2-terminal amino acids are involved in apoptotic death control. This selective inhibition was followed by an early, overall but also target-specific inhibition of ubiquitination and by an activation and stabilization of poly(ADP-ribose) polymerase (PARP) that occurs through inhibition of ubiquitination of its cleaved form (85 kDa). Interestingly, caspases-3 and -7 were not activated following irradiation, further suggesting that PARP cleavage may be regulated by an N-end rule pathway in a caspase-independent manner. These results highly suggest involvement of this subset of the ubiquitin system in the apoptotic death control and in the specific regulation of PARP activity.

Purpose : Prospective investigation of spontaneous and in vitro radiation-induced apoptosis to predict early response to palliative radiotherapy in patients with non-Hodgkin's lymphomas. Patients and methods : Fine-needle sampling was performed in 28 tumor sites (26 patients) and yielded adequate cell numbers in 27 cases. Apoptotic cells were counted by fluorescence microscopy immediately after sampling and after 24-h culture (spontaneous apoptosis) and 24 h after 2- and 10-Gy in vitro irradiation (radiation-induced apoptosis). Early response to low-dose in vivo radiotherapy (mostly 4 Gy in two fractions over 3 days) was evaluated 15 days after treatment. Results : The tumor response rates at 15 days were 11 (39%) complete responses, nine (32%) responses of greater than 50% reduction in volume, six (21%) responses of less than 50% reduction in volume and two (7%) cases of no response. Tumors achieving complete or major response after in vivo irradiation had higher percentages of apoptotic cells after in vitro irradiation, while no significant differences in terms of spontaneous apoptosis were observed between responders and non-responders. Conclusion : Spontaneous and in vitro radiation-induced apoptosis can be easily and quickly assessed on cells obtained by fine-needle sampling of non-Hodgkin's lymphoma lesions. The present results suggest that in vitro radiation-induced apoptosis could be used as a predictive assay of early response to low-dose in vivo irradiation in patients with non-Hodgkin's lymphomas.

Apoptosis, related to a naturally-occurring or programmed cellular death process, can be physiologically or exogenously induced. In vertebrate cells undergoing apoptosis, initiated by any of these ways, one of the numerous biochemical changes is an endogenous endonuclease activation that cleaves the chromatin DNA into oligonucleosome-sized 'ladder' fragments. In the present study we show that in parallel to chromatin DNA cleavage, ribosomal RNA is lost in gamma-ray-mediated apoptotic human lymphocytes. We demonstrate that 28S rRNA gene transcription is induced early (15 min) after irradiation, followed by a selective disappearance in apoptotic cells only. The fact that newly synthesized rRNA turns over at the same rate in irradiated and untreated cell fractions, highly suggests that the observed loss of 28S rRNA in the apoptotic cell fraction at the ribosome level is due to degradation occurring at a late stage of the apoptotic death process. These results suggest that, in addition to first-stage apoptosis-associated rDNA gene activation, cellular self-destruction at late stages is associated with processes occurring simultaneously at the ribosome level involving an endogenous RNase-like activity, and at the chromatin level involving DNA-nuclease activity.

The ability of a “vital” dye, acridine orange (AO), to intercalate into the DNA of living cells was investigated by quantitative intensified fluorescence microscopy and digital imaging under various conditions of dye concentration, excitation light intensity, and ionic concentration. Our results demonstrate that the bulk of chromatin DNA is packed in a way that does not allow AO intercalation. At low dye concentrations and very low levels of light intensity, the only fluorescent structures observed inside the nucleus are the nucleoli. This nonpermissive state of the chromatin appears to be a characteristic feature of the nucleus in living cells. AO intercalation into DNA can be mediated by raising the nuclear Na + concentration. This was achieved here by using a cation carrier, monensin, a procedure which permits the avoidance of cell permeabilization. Furthermore, we show that the discharge of lysosomal enzymes in the living cell, via a targeted photodynamic reaction which occurs at high levels of light intensity, can also release the constraints which impede dye intercalation into nuclear DNA. In conclusion, studies carried out under conditions where intercalative dyes such as AO are able to stain the nuclear DNA have to be interpreted with caution and do not provide direct evidence on the structural state of native chromatin. The molecular origin of the absence of AO intercalation in chromatin of the living cell is discussed with regard to the restrained uncoiling of the double helix which is required for dye intercalation.

With the aim of obtaining new antitumor drugs more active than previously described 5, 8-quinazolinediones, a series of dimers of 5, 8-quinazolinediones linked in the 4-position by a simple or a substituted χ, ω-diaminopolymethylenic chain was studied. The structure-activity relationships of these compounds are discussed as functions of the length of the chain, presence or absence of other functional groups, nature of these functional groups, position of the chain and nature of the substituents in the 6 and (or) 7-positions. When bis (5, 8-quinazolinediones) were substituted in the 6-position with a methoxyl group, the dimerization showed a variable effect on cytotoxicity toward L 1210 leukemia cells. Bis [4-amino-bis-6, 7 (1-aziridinyl)-5, 8-quinazolinediones] which exhibited high cytotoxic activity (IC50 0.0037 to 0.018μm) were further screened in vivo for activity against murine P388 leukemia. Antitumor activity was increased by the dimerization of the molecule. The most potent compound bears an additional tertiary amino function on the chain.