USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora-A kinase.

A large number of patients are resistant to taxane-based chemotherapy. Functional mitotic checkpoints are essential for taxane sensitivity. Thus, mitotic regulators are potential markers for therapy response and could be targeted for anticancer therapy. In this study, we identified a novel function of ubiquitin (Ub)-specific processing protease-7 (USP7) that interacts and cooperates with protein death domain-associated protein (Daxx) in the regulation of mitosis and taxane resistance. Depletion of USP7 impairs mitotic progression, stabilizes cyclin B and reduces stability of the mitotic E3 Ub ligase, checkpoint with forkhead and Ring-finger (CHFR). Consequently, cells with depleted USP7 accumulate Aurora-A kinase, a CHFR substrate, thus elevating multipolar mitoses. We further show that these effects are independent of the USP7 substrate p53. Thus, USP7 and Daxx are necessary to regulate proper execution of mitosis, partially via regulation of CHFR and Aurora-A kinase stability. Results from colony formation assay, in silico analysis across the NCI60 platform and in breast cancer patients suggest that USP7 levels inversely correlate with response to taxanes, pointing at the USP7 protein as a potential predictive factor for taxane response in cancer patients. In addition, we demonstrated that inhibition of Aurora-A attenuates USP7-mediated taxane resistance, suggesting that combinatorial drug regimens of Taxol and Aurora-A inhibitors may improve the outcome of chemotherapy response in cancer patients resistant to taxane treatment. Finally, our study offers novel insights on USP7 inhibition as cancer therapy.

CHEK2 genomic and proteomic analyses reveal genetic inactivation or endogenous activation across the 60 cell lines of the US National Cancer Institute.

CHEK2 encodes a serine/threonine kinase (Chk2) activated by ATM in response to DNA double-strand breaks. On the one hand, CHEK2 has been described as a tumor suppressor with proapoptotic, cell-cycle checkpoint and mitotic functions. On the other hand, Chk2 is also commonly activated (phosphorylated at T68) in cancers and precancerous lesions. Here, we report an extensive characterization of CHEK2 across the panel of 60 established cancer cell lines from the NCI Anticancer Screen (the NCI-60) using genomic and proteomic analyses, including exon-specific mRNA expression, DNA copy-number variation (CNV) by aCGH, exome sequencing, as well as western blot analyses for total and activated (pT68-Chk2) Chk2. We show that the high heterogeneity of Chk2 levels in cancer cells is primarily due to its inactivation (owing to low gene expression, alternative splicing, point mutations, copy-number alterations and premature truncation) or reduction of protein levels. Moreover, we observe that a significant percentage of cancer cells (12% of the NCI-60 and HeLa cells) show high endogenous Chk2 activation, which is always associated with p53 inactivation, and which is accompanied by downregulation of the Fanconi anemia and homologous recombination pathways. We also report the presence of activated Chk2 (pT68-Chk2) along with histone γ-H2AX in centrosomes.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

c-myc Down-regulation in suramin-treated HL60 cells precedes growth inhibition but does not trigger differentiation.

Down-regulation of c-myc mRNA expression is linked to growth arrest and the state of differentiation of hematopoietic cells. We showed that treatment of HL60 cells with suramin results in a rapid reduction of c-myc expression followed by an inhibition of cell growth. Both c-myc mRNA and protein levels decreased by day 1 of treatment, and, by day 4, only 10% of control c-myc protein levels were detected. In contrast to retinoic acid, dimethyl sulfoxide, and 12-O-tetradecanoylphorbol-13-acetate treatment, however, exposure of HL60 cells to suramin did not result in the induction of differentiation. These results demonstrate that suramin modulates c-myc levels in HL60 cells and that the down-regulation of c-myc is not sufficient to trigger differentiation toward either granulocytic or monocytic lineages.

Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms.

Expression of the vascular permeability factor/vascular endothelial growth factor (VEGPF) gene was investigated in human central nervous system (CNS) neoplasms and normal brain. Adsorption of capillary permeability activity from human glioblastoma multiforme (GBM) cell conditioned medium and GBM cyst fluids by anti-VEGPF antibodies demonstrated that VEGPF is secreted by GBM cells and is present in sufficient quantities in vivo to induce vascular permeability. Cloning and sequencing of polymerase chain reaction-amplified GBM and normal brain cDNA demonstrated three forms of the VEGPF coding region (567, 495, and 363 nucleotides), corresponding to mature polypeptides of 189, 165, and 121 amino acids, respectively. VEGPF mRNA levels in CNS tumors vs. normal brain were investigated by the RNase protection assay. Significant elevation of VEGPF gene expression was observed in 81% (22/27) of the highly vascular and edema-associated CNS neoplasms (6/8 GBM, 8/8 capillary hemangioblastomas, 6/7 meningiomas, and 2/4 cerebral metastases). In contrast, only 13% (2/15) of those CNS tumors that are not commonly associated with significant neovascularity or cerebral edema (2/10 pituitary adenomas and 0/5 nonastrocytic gliomas) had significantly increased levels of VEGPF mRNA. The relative abundance of the forms of VEGPF mRNA was consistent in tumor and normal brain: VEGPF495 > VEGPF363 > VEGPF567. In situ hybridization confirmed the presence of VEGPF mRNA in tumor cells and its increased abundance in capillary hemangioblastomas. Our results suggest a significant role for VEGPF in the development of CNS tumor neovascularity and peritumoral edema.

Directionality and further mapping of varicella zoster virus transcripts.

Our laboratory previously identified and preliminarily mapped 58 viral RNA transcripts in varicella zoster virus (VZV) infected cells (Ostrove et al., 1985). This study was initiated to more precisely map these transcripts, to identify additional transcripts, and to determine transcript directionality. To accomplish this, 32 overlapping BamHI, EcoRI, and SmaI fragments representing 99.7% of the genome were cloned into pGEM-2, a plasmid which contains a multiple cloning site flanked by SP6 and T7 RNA polymerase promoters. Each of these clones was used to produce 32P-labeled double-stranded DNA probes to detect transcripts homologous to either strand of the VZV insert, and single-stranded [32P]RNA probes in order to detect RNAs of either polarity. These probes were hybridized to Northern blots of VZV-infected cell RNA. In all, 77 RNAs were detected with both DNA and RNA probes. The direction of transcription and localization of 57 of the 58 previously identified RNAs and of 20 newly recognised abundant transcripts were determined. Thirty-three additional low-abundance transcripts were detected only by the relatively more sensitive RNA probes. A map indicating the directionality and approximate locations of the abundant VZV transcripts was constructed.

Inversion and circularization of the varicella-zoster virus genome.

The genome of varicella-zoster virus (VZV) is a linear, double-stranded molecule of DNA composed of a long (L) region covalently linked to a short (S) region. The S region is capable of inverting relative to a fixed orientation of the L region, giving rise to two equimolar populations. We have investigated other forms of the VZV genome which are present in infected cells and packaged into nucleocapsids. That a small proportion of nucleocapsid DNA molecules also possess inverted L regions has been verified by the identification of submolar restriction fragments corresponding to novel joints and novel ends generated by such an inversion. The presence of circular molecules has been investigated by agarose gel electrophoresis. Bands corresponding to circular forms were present in small amounts in both VZV-infected cell DNA and nucleocapsid DNA. Southern blot analysis verified that these bands contained VZV sequences. We therefore conclude that the VZV genome may occasionally contain an inverted L region or exist in a circular configuration.