Development and validation of an RT-qPCR assay for rapid detection and quantification of hepatitis C virus RNA for routine testing in Moroccan clinical specimens.

A one-step reverse transcription quantitative PCR (RT-qPCR) assay in combination with rapid RNA extraction was evaluated for routine testing of hepatitis C virus (HCV) RNA. Specific primers and probes were designed for the detection of a 150 bp sequence located in the 5'untranslated region (5'UTR) of HCV RNA. The target sequence was selected as the most conserved region between the six known HCV subtype sequences following an alignment. The assay was able to quantify a dynamic linear range of 108 to 101 plasmid copies/reaction (r2 = 0.98) containing the target sequence. Two copies of this HCV plasmid corresponds to one international unit (IU) measured using a standard obtained by serial dilutions of the World Health Organization (WHO) standard. The detection limit of the assay was about 10 IU/mL of HCV RNA (20 copies/mL) in plasma samples. The assay was comparable to Cobas AmpliPrep/Cobas TaqMan® HCV Test, v2.0 Quantitative assay (Roche Molecular Systems, Inc., Branchburg, NJ) with correlation coefficient r2 = 0.98. The present assay could be completed within 3 hours from RNA extraction to data analysis of at least 30 plasma samples. Our test provides sufficient sensitivity, specificity, and reproducibility and proved to be fast, labor-saving, and cost-effective. Indeed, our system will definitely allow low-income countries to monitor accurately this viral infection and to efficiently treat their infected patients.

A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation.

BACKGROUND: The mammalian DNA-damage response (DDR) has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs), mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. METHODS: In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+) with the MDM2 antagonist, Nutlin-3. RESULTS: Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37). Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. CONCLUSION: To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

Role of ubiquitination in the DNA damage response: proteomic analysis to identify new DNA-damage-induced ubiquitinated proteins.

The DDR (DNA damage response) is a signalling transduction cascade utilizing many forms of post-translation modification of proteins, including phosphorylation and ubiquitination. The well-known function of ubiquitination is to target proteins for proteasomal degradation; however, it is also involved in the regulation of protein function. The present review describes how ubiquitination regulates the function of certain proteins involved in DDR, in particular FANCD2 (Fanconi's anaemia complementation group D2) and PCNA (proliferating-cell nuclear antigen). Also, the proteomic methods currently used to identify new ubiquitinated proteins in response to DNA damage, including the advantages of using the UBD (ubiquitin-binding domain) beads to purify the ubiquitinated proteins, are considered.

Design, synthesis, chemical characterization, biological evaluation, and docking study of new 1,3,4-oxadiazole homonucleoside analogs.

Herein, we report the synthetic strategies and characterization of some novel 1,3,4-oxadiazole homonucleoside analogs that are relevant to potential antitumor and cytotoxic activities. The structure of all compounds is confirmed using various spectroscopic methods such as 1H-NMR, 13C-NMR, HRMS, and FTIR. These compounds were evaluated against three human cancer cell lines (MCF-7, SKBR3, and HL60 Cell Line). Preliminary investigations showed that the cytotoxic activity was markedly dependent on the nucleobase. Introduction of 5-Iodouracil 4g and theobromine 6b proved to be extremely beneficial even they were more potent than the reference drug (DOX). Also, the synthesized compounds were tested for their antiviral activities against the human varicella-zoster virus (VZV). The product 4h was (6-azauracil derivative) more potent to the reference (acyclovir) against the deficient TK - VZV strain by about 2-fold. Finally, molecular docking suggested that the anticancer activities of compounds 6b and 4g mediated by inhibiting dual proteins EGFR/HER2 with low micromolar inhibition constant Ki range. The 1,3,4-oxadiazole homonucleosides showed a strong affinity to binding sites of target proteins by forming H-bond, carbon-hydrogen bond, Pi-anion, Pi-sulfur, Pi-sigma, alkyl, and Pi-alkyl interactions.

Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the first study using the specific combination of RPL30 and RPL37 as reference genes for an accurate HER2 gene quantification in FFPE biopsy samples. Although further clinical validation regarding evolution and therapeutic response using RT-qPCR for the quantification of HER2 expression are still needed, the present study constitutes definitely a factual element that the RT-qPCR based assay may constitute a valid complementary test to accurately measure HER2 expression for a better treatment orientation.

Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer.

Evaluation of Combined Quantification of PCA3 and AMACR Gene Expression for Molecular Diagnosis of Prostate Cancer in Moroccan Patients by RT-qPCR

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1.

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.

Evaluation of a novel multiplex RT-qPCR assay for the quantification of leukemia-associated BCR-ABL1 translocation.

Although monitoring of BCR-ABL1 translocation has become an established practice in the management of chronic myeloid leukemia (CML), the detection limit of the BCR-ABL1 transcripts needs more standardization. The aim of the present study was to evaluate the clinical performances of a novel assay for the quantification of BCR-ABL1 fusion transcripts (e13a2 and e14a2) and ABL1 in a single reaction. This assay is based on the real-time reverse transcription polymerase chain reaction (RT-qPCR) in multiplex format. In a retrospective comparative clinical study performed in a reference laboratory, RNA was extracted from 48 CML patient blood samples with various BCR-ABL1/ABL1 ratios and RT-qPCR was performed using either MAScIR assay or the RT-qPCR simplex reference assay used in routine clinical testing. The comparative clinical results showed high qualitative and quantitative concordance (correlation coefficient >0.95) between MAScIR and the reference assays. The present study illustrates the utility of MAScIR assay as a sensitive, rapid, and cost-effective quantitative device to monitor the BCR-ABL1 ratios by RT-qPCR on whole blood of diagnosed Philadelphia chromosome-positive (Ph+) leukemia patients. This test could be used as an aid in the assessment of molecular response to available treatments.

ATM-dependent phosphorylation of heterogeneous nuclear ribonucleoprotein K promotes p53 transcriptional activation in response to DNA damage.

Previous work has established that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is stabilized in an ATM-dependent manner in response to DNA damage and acts as a cofactor for p53-mediated transcription. Here, we show that in response to DNA damage caused by ionizing radiation, hnRNP K is phosphorylated in an ATM-dependent manner. Furthermore, our data indicate that ATM-dependent hnRNP K phosphorylation is required for its stabilization and its function as a p53 transcriptional cofactor in response to DNA damage. These findings thereby establish hnRNP K as an ATM target and help define how ATM orchestrates p53-dependent transcriptional responses in response to genotoxic stress.

MDM2-dependent downregulation of p21 and hnRNP K provides a switch between apoptosis and growth arrest induced by pharmacologically activated p53.

We have previously identified the p53-reactivating compound RITA in a cell-based screen. Here, using microarray analysis, we show that the global transcriptional response of tumor cells to RITA is p53 dependent. Pathway analysis revealed induction of the p53 apoptosis pathway, consistent with apoptosis being the major response to RITA in cancer cells. We uncovered that MDM2 released from p53 by RITA promotes degradation of p21 and the p53 cofactor hnRNP K, required for p21 transcription. Functional studies revealed MDM2-dependent inhibition of p21 as a key switch regulating cell fate decisions upon p53 reactivation. Our results emphasize the utility of targeting wild-type p53 protein itself as a promising approach for anticancer therapy.

hnRNP K: an HDM2 target and transcriptional coactivator of p53 in response to DNA damage.

In response to DNA damage, mammalian cells trigger the p53-dependent transcriptional induction of factors that regulate DNA repair, cell-cycle progression, or cell survival. Through differential proteomics, we identify heterogeneous nuclear ribonucleoprotein K (hnRNP K) as being rapidly induced by DNA damage in a manner that requires the DNA-damage signaling kinases ATM or ATR. Induction of hnRNP K ensues through the inhibition of its ubiquitin-dependent proteasomal degradation mediated by the ubiquitin E3 ligase HDM2/MDM2. Strikingly, hnRNP K depletion abrogates transcriptional induction of p53 target genes and causes defects in DNA-damage-induced cell-cycle-checkpoint arrests. Furthermore, in response to DNA damage, p53 and hnRNP K are recruited to the promoters of p53-responsive genes in a mutually dependent manner. These findings establish hnRNP K as a new HDM2 target and show that, by serving as a cofactor for p53, hnRNP K plays key roles in coordinating transcriptional responses to DNA damage.

The structure of HIV-1 genomic RNA in the gp120 gene determines a recombination hot spot in vivo.

By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.

Evidence for a mechanism of recombination during reverse transcription dependent on the structure of the acceptor RNA.

Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination.