A 'DNA replication' signature of progression and negative outcome in colorectal cancer.

Colorectal cancer is one of the most frequent cancers worldwide. As the tumor-node-metastasis (TNM) staging classification does not allow to predict the survival of patients in many cases, additional prognostic factors are needed to better forecast their outcome. Genes involved in DNA replication may represent an underexplored source of such prognostic markers. Indeed, accidents during DNA replication can trigger 'replicative stress', one of the main features of cancer from earlier stages onward. In this study, we assessed the expression of 47 'DNA replication' genes in primary tumors and adjacent normal tissues from a homogeneous series of 74 patients. We found that genes coding for translesional (TLS) DNA polymerases, initiation of DNA replication, S-phase signaling and protection of replication forks were significantly deregulated in tumors. We also observed that the overexpression of either the MCM7 helicase or the TLS DNA polymerase POLQ (if also associated with a concomitant overexpression of firing genes) was significantly related to poor patient survival. Our data suggest the existence of a 'DNA replication signature' that might represent a source of new prognostic markers. Such a signature could help in understanding the molecular mechanisms underlying tumor progression in colorectal cancer patients.

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery.

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.

Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells.

Following prolonged mitotic spindle disruption by microtubule poisons, mammalian cells delay their entry into anaphase, then progressively slip out of mitosis and become tetraploid. Normal cells then stop cycling before S-phase onset, but the mechanisms underlying this arrest are still unclear. Here we show that a double block prevents endo-reduplication. First, cells that exit mitosis without a functional microtubule network are driven toward G0. Reconstitution of the network unmasks a second block that relies on DNA double-strand breaks occurring early in the G1 phase that follows the mitotic block. We propose that a stress signal elicited upon mitotic impairment triggers breakage, which couples the leaky spindle checkpoint to the stringent DNA damage response. Consistent with this finding, cells defective for the damage response continue cycling and acquire, within a single cell cycle, both chromosome rearrangements and abnormal chromosome numbers that remarkably mimic the complex genetic hallmark of tumorigenesis.

Characterization of promoter regulatory elements involved in downexpression of the DNA polymerase kappa in colorectal cancer.

The low-fidelity DNA polymerases thought to be specialized in DNA damage processing are frequently misregulated in cancers. We show here that DNA polymerase kappa (polkappa), prone to replicate across oxidative and aromatic adducts and known to function in nucleotide excision repair (NER), is downregulated in colorectal tumour biopsies. Contrary to the replicative poldelta and polalpha, for which only activating domains were described, we identified an upstream 465-bp-long repressor region in the promoter of POLK. We also found an activating 237-bp region that includes stimulating protein-1 (SP1) and cyclic AMP-responsive element (CRE)-binding sites. Mutations at one CRE-binding site led to a dramatic 80% decrease in promoter activity. Alterations of the SP1-binding site also affected, to a lesser extent, the transcription. Gel shift assays confirmed the role played by CRE/SP1 recognition sequences. Moreover, ectopic expression of SP1 or CRE-binding protein (CREB) protein favoured polkappa transcription. Finally, we found that polkappa downexpression in colorectal biopsies correlated with a decreased level of CREB and SP1 transcripts. This work shows that the promoter of POLK is cis-controlled and suggests that silencing of CREB and SP1 proteins could contribute to downregulation of this repair polymerase in colorectal tumours.

Modulation of cellular response to cisplatin by a novel inhibitor of DNA polymerase beta.

DNA polymerase beta (Pol beta) is an error-prone enzyme whose up-regulation has been shown to be a genetic instability enhancer as well as a contributor to cisplatin resistance in tumor cells. In this work, we describe the isolation of new Pol beta inhibitors after high throughput screening of 8448 semipurified natural extracts. In vitro, the selected molecules affect specifically Pol beta-mediated DNA synthesis compared with replicative extracts from cell nuclei. One of them, masticadienonic acid (MA), is particularly attractive because it perturbs neither the activity of the purified replicative Pol delta nor that of nuclear HeLa cell extracts. With an IC50 value of 8 microM, MA is the most potent of the Pol beta inhibitors found so far. Docking simulation revealed that this molecule could substitute for single-strand DNA in the binding site of Pol beta by binding Lys35, Lys68, and Lys60, which are the main residues involved in the interaction Pol beta/single-strand DNA. Selected inhibitors also affect the Pol beta-mediated translesion synthesis (TLS) across cisplatin adducts; MA was still the most efficient. Therefore, masticadienonic acid sensitized the cisplatin-resistant 2008C13*5.25 human tumor cells. Our data suggest that molecules such as masticadienonic acid could be suitable in conjunction with cisplatin to enhance anticancer treatments.

Antitumor activity of 2',3'-dideoxycytidine nucleotide analog against tumors up-regulating DNA polymerase beta.

DNA polymerase beta (Pol beta), an error-prone DNA-synthesizing enzyme tightly down-regulated in healthy somatic cells, has been shown to be overexpressed in many human tumors. In this study, we show that treatment with the 2',3'-dideoxycytidine (ddC) nucleoside analog inhibited in vitro and in vivo the proliferation of Pol beta-transfected B16 melanoma cells, which up-regulate Pol beta compared with control isogenic cells. The administration of ddC also increased specifically the survival of mice bearing Pol beta-overexpressing B16 melanoma. When the phosphorylated form of ddC was electrotransfered into Pol beta-transfected melanoma, the cell growth inhibition was strengthened, strongly suggesting that the cytotoxic effect results from incorporation of the chain terminator into DNA. Using in vitro single- and double-stranded DNA synthesis assays, we demonstrated that excess Pol beta perturbs the replicative machinery, favors ddC-TP incorporation into DNA, and consequently promotes chain termination. Therefore, the use of chain terminator anticancer agents could be suitable for the treatment of tumors with a high level of Pol beta.

A new role for hypoxia in tumor progression: induction of fragile site triggering genomic rearrangements and formation of complex DMs and HSRs.

Genome rearrangements including gene amplification are frequent properties of tumor cells, but how they are related to the tumor microenvironment is unknown. Here, we report direct evidence for a causal relationship between hypoxia, induction of fragile sites, and gene amplification. Recently, we showed that breaks at fragile sites initiate intrachromosomal amplification. We demonstrate here that hypoxia is a potent fragile site inducer and that, like fragile sites inducing drugs, it drives fusion of double minutes (DMs) and their targeted reintegration into chromosomal fragile sites, generating homogeneously staining regions (HSRs). This pathway operates efficiently for DMs bearing different sequences, suggesting a model of hypoxia-driven formation of the HSRs containing nonsyntenic sequences frequently observed in solid tumors.

Interstitial deletions and intrachromosomal amplification initiated from a double-strand break targeted to a mammalian chromosome.

Interstitial deletions of tumour suppressor genes and amplification of oncogenes are two major manifestations of chromosomal instability in tumour cells. The development of model systems allowing the study of the events triggering these processes is of major clinical importance. Using the properties of the I-SceI nuclease to introduce a localized double-strand break (DSB) in a mammalian chromosome carrying its target sequence, we demonstrate here that both types of mutations can be initiated by non-conservative DSB repair pathways. In our system, I-SceI activity dissociates a transfected gpt gene from its promoter, allowing the isolation of gpt- clones. Our results show that intrachromatid single-strand annealing events occur frequently, giving rise to interstitial deletions not accompanied by other chromosomal rearrangements. We also observed that, when present in the cells, extrachromosomal DNA molecules are integrated preferentially at the broken locus. Taking advantage of the insertion of the I-SceI recognition sequence telomeric to and close to the dihydrofolate reductase gene, we show that a less frequent outcome of I-SceI activity is the initiation of cycles of intrachromosomal amplification of this marker, from breaks at a site merging with the enzyme target.

Masked diabetic mastoiditis.

Seven cases of elderly diabetics suffering from ths so-called 'malignant external otitis' are presented. Chandler (1968) and others consider that this disease is localized to the external ear canal. We do not agree and believe that it is due to a masked middle-ear infection extending through the tympano-mastoid cells to the mastoid process, sparing the antrum and bulging into the floor of the external meatus. In time, extension to petrous apex results in petrositis and a fatal end in many cases.