Evaluation of the Abbott RealTime quantitative CMV and EBV assays using the maxCycle protocol in a laboratory automation context.

Real-time PCR are often used for the diagnosis and monitoring of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections in susceptible populations. In this context, we evaluated the analytical performances of the Abbott RealTime CMV/EBV maxCycle protocol automated on the m2000 platform (Abbott). It was compared to our routinely-used procedure consisting of a NucleoMag® DNA extraction automated on a STARlet platform followed by manually processed CMV and EBV quantitative real-time PCR (Diagenode). In this study, we showed that both EBV assays exhibited a similar sensitivity but with a better precision for the EBV Abbott RealTime assay. For the CMV performances, the Abbott assay was more sensitive and more precise than our routine method. The use of WHO International Standards also indicated a slight underestimation of the viral loads (-0.25 log10 IU/mL and -0.21 log10 IU/mL for CMV and EBV assays respectively) while these were rather overestimated with the Starlet/Diagenode method (0.48 log10 IU/mL and 0.19 log10 IU/mL for CMV and EBV assays respectively). These trends were confirmed using relevant whole-blood clinical samples and external quality controls. The workflows were also compared and we highlighted a significant technician hands-on time reduction (-63%) using the Abbott CMV/EBV maxCycle automated protocol.

Chromatin redistribution of the DEK oncoprotein represses hTERT transcription in leukemias.

Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis.

Casein kinase 2 inhibition modulates the DNA damage response but fails to radiosensitize malignant glioma cells.

Inhibitors of casein kinase 2 (CK2), a regulator of cell proliferation and mediator of the DNA damage response, are being evaluated in clinical trials for the treatment of cancers. Apigenin was capable of inhibiting the activation of CK2 following γ irradiation in LN18 and U87 malignant glioma cells. Apigenin and siRNA-mediated CK2 protein depletion further inhibited NF-κB activation and altered the Tyr68 phosphorylation of Chk2 kinase, a DNA damage response checkpoint kinase, following irradiation. However, CK2 inhibition did not decrease the ability of these glioma cells to repair double-strand DNA breaks, as assessed by COMET assays and γ-H2Ax staining. Likewise, apigenin and siRNA-induced depletion of CK2 failed to sensitize glioma cells to the cytotoxic effect of 2 to 10 G-rays of γ irradiation, as assessed by clonogenic assays. These results contrast with those found in other cancer types, and urge to prudence regarding the inclusion of malignant glioma patients in clinical trials that assess the radiosensitizing role of CK2 inhibitors in solid cancers.

HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells.

Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression.

Human glioblastoma-initiating cells invade specifically the subventricular zones and olfactory bulbs of mice after striatal injection.

In patients with glioblastoma multiforme, recurrence is the rule despite continuous advances in surgery, radiotherapy and chemotherapy. Within these malignant gliomas, glioblastoma stem cells or initiating cells have been recently described, and they were shown to be specifically involved in experimental tumorigenesis. In this study, we show that some human glioblastoma cells injected into the striatum of immunodeficient nude mice exhibit a tropism for the subventricular zones. There and similarily to neurogenic stem cells, these subventricular glioblastoma cells were then able to migrate toward the olfactory bulbs. Finally, the glioblastoma cells isolated from the adult mouse subventricular zones and olfactory bulbs display high tumorigenicity when secondary injected in a new mouse brain. Together, these data suggest that neurogenic zones could be a reservoir for particular cancer-initiating cells.

Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias.

OBJECTIVE: Telomeres are protected by tightly regulated factors and elongated by telomerase. Short and/or deprotected chromosomes are recombinogenic and thereby cancer prone. MATERIALS AND METHODS: Together with the quantification of telomerase activity (TA), measuring telomere length (TL) and expression of the genes that govern telomere protection and elongation are useful for assessing telomere homeostasis. RESULTS: By these means we demonstrate that TL, hTERT, and TA are in the order acute myelogenous leukemia (AML) > T-cell acute lymphoblastic leukemia (T-ALL) > B-cell acute lymphoblastic leukemia (B-ALL) > T-ALL > AML, and B-ALL > AML > T-ALL. AML0 and AML3 display the lowest amounts of hTERT transcripts, and ALL and AML cells with cytogenetic abnormalities possess the shortest telomeres. hTERT expression includes phenotype-specific RNA maturation and correlates with TA but not with TL. A wide ratio of TA to hTERT expression between leukemia subtypes suggests phenotype-specific hTERT post-transcriptional deregulations. B- and T-ALL overexpress Ku70 and Pinx1, T-ALL PTOP and RAP1, and B-ALL TRF2, the expression of which is significantly higher in cases with abnormal karyotype. hTERT transcription and TL correlate with response to intensive chemotherapy, and hTERT and RAD50 are independent prognostic factors for survival. CONCLUSIONS: Each leukemia subtype possesses specific telomere dysregulations that rely on phenotype, karyotype, response to treatment, and survival.