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.

The p400/Tip60 ratio is critical for colorectal cancer cell proliferation through DNA damage response pathways.

The Tip60 histone acetyltransferase belongs to a multimolecular complex that contains many chromatin remodeling enzymes including the ATPase p400, a protein involved in nucleosomal incorporation of specific histone variants and that can directly or indirectly repress some Tip60-dependent pathways. Tip60 activity is critical for the cellular response to DNA damage and is affected during cancer progression. Here, we found that the ratio between Tip60 and p400 mRNAs is affected in most colorectal carcinoma. Strikingly, reversing the p400/Tip60 imbalance by Tip60 overexpression or the use of siRNAs resulted in increased apoptosis and decreased proliferation of colon-cancer-derived cells, suggesting that this ratio defect is important for cancer progression. Furthermore, we demonstrate that the p400/Tip60 ratio controls the oncogene-induced DNA damage response, a known anticancer barrier. Finally, we found that it is also critical for the response to 5-fluorouracil, a first-line treatment against colon cancer. Together, our data indicate that the p400/Tip60 ratio is critical for colon cancer cells proliferation and response to therapeutic drugs through the control of stress-response pathways.

Novel evidences for a tumor suppressor role of Rev3, the catalytic subunit of Pol zeta.

Cell cycle checkpoints and DNA repair act in concert to ensure DNA integrity during perturbation of normal replication or in response to genotoxic agents. Deficiencies in these protective mechanisms can lead to cellular transformation and ultimately tumorigenesis. Here we focused on Rev3, the catalytic subunit of the low-fidelity DNA repair polymerase zeta. Rev3 is believed to play a role in double-strand break (DSB)-induced DNA repair by homologous recombination. In line with this hypothesis, we show the accumulation of chromatin-bound Rev3 protein in late S-G2 of untreated cells and in response to clastogenic DNA damage as well as an gamma-H2AX accumulation in Rev3-depleted cells. Moreover, serine 995 of Rev3 is in vitro phosphorylated by the DSB-inducible checkpoint kinase, Chk2. Our data also disclose a significant reduction of rev3 gene expression in 74 colon carcinomas when compared to the normal adjacent tissues. This reduced expression is independent of the carcinoma stages, suggesting that the downregulation of rev3 might have occurred early during tumorigenesis.

Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation.

Preclinical studies have demonstrated that the chemotherapeutic action of oxaliplatin, a third generation platinum derivative, is improved when combined with cetuximab, a monoclonal antibody inhibitor of epidermal growth factor receptors. To explore the mechanism of this synergistic benefit, we used HCT-8 and HCT-116, two human colon cancer cell lines, respectively, responsive and non-responsive to the oxaliplatin/cetuximab combination. We examined the effect of drug exposure on glutathione-S-transferase-mediated oxaliplatin detoxification, DNA-platinum adducts formation, cell cycle distribution, apoptosis, and the expression of multiple targets involved in DNA replication, recombination, and repair. The major changes we found in HCT-8 were a stimulation of oxaliplatin-DNA adduct formation associated with reduced expression of the key enzyme (excision repair cross complementation group1: ERCC1) in the key repair process of oxaliplatin-DNA platinum adduct, the nucleotide excision repair (NER), both at the mRNA and protein levels. We also observed a reduced expression of factors involved in DNA replication initiation, which correlates with an enrichment of cells in the G1 phase of the cell cycle as well as an acceleration of apoptosis. None of these changes occurred in the non-responsive HCT-116 cell that we used as a negative control. These findings support the fact that cetuximab potentiates the oxaliplatin-mediated cytotoxic effect as the result of inhibition of NER and also DNA replication initiation.

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.

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.

Cisplatin and UV radiation induce activation of the stress-activated protein kinase p38gamma in human melanoma cells.

The p38 alpha mitogen-activated protein kinase has been implicated in the cellular response to genotoxic agents. Here we show that another p38 family member is also activated in response to cisplatin exposure in human melanoma cells. We identified this isoform as p38gamma based on its recognition by specific antibodies and because, in contrast to p38alpha, its activity was not affected by SB203580. We also found that etoposide caused a much more discrete phosphorylation of both p38alpha and p38gamma than either cisplatin or UV treatment. These results indicate that genotoxic stresses activate several p38 isoforms whose implication in the cellular response might depend on the type of DNA damage.

Regulation by transforming growth factor-beta 1 of G1 cyclin-dependent kinases in human retinal epithelial cells.

Transforming growth factor beta (TGFbeta) is a potent inhibitor of epithelial cell proliferation, delaying or arresting cell cycle progression in mid-late G1. In long-term life span cells this growth inhibitory action has been attributed to regulatory events on both the levels and activities of G1 cyclin-dependent kinases (CDKs). CDK inhibitors have been shown to play important role in the TGFbeta-induced inhibition of G1 CDKs. In this work, we have investigated the effect of TGFbeta1 on both cell proliferation and G1 CDK activities in primary cultures of human retinal pigment epithelial (RPE) cells. We show that TGFbeta1 exerts a partial inhibitory effect on RPE cell proliferation by causing a significant increase of the RPE cell number in G1. TGFbeta1 induces an up-regulation of the CDK inhibitor p15(INK4B)with its subsequent association to CDK4, and a decline in CDK4 protein level. In parallel, we have observed a decline of p27(KIP1)associated to CDK4 and a significant increase of the inhibitor associated to CDK2. Finally, we show that TGFbeta1 reduces both CDK4 and CDK2 enzymatic activities. The fact that TGFbeta exerts only partial inhibitions on G1 CDKs and cell cycle progression in RPE cells suggests a propensity of these cells to escape from the anti-proliferative action of the cytokine, a phenomenon which could be reinforced during the development of proliferative vitreoretinopathy.

Differential expression of G1 cyclins and cyclin-dependent kinase inhibitors in normal and transformed melanocytes.

PURPOSE: To investigate the levels of the different regulatory proteins involved in the G1 progression and G1/S transition in normal and transformed human choroidal melanocytes (CM). METHODS: Three choroidal melanoma cell lines and three CM cultures were used. The purity of the CM cultures was assessed by different approaches, including morphologic study, specific immunostaining, cell proliferation behavior, and transforming growth factor-beta1 responsiveness. The cell cycle protein levels were evaluated by specific immunoblotting of total extracts obtained from the different cell lines. RESULTS: Alterations were observed in the expression of cylins D1 and E in the transformed cells, whereas the amounts of the cyclin-dependent kinases (CDKs) CDK2 and CDK4 were almost identical in both cell types. Although the expression of cyclin H was slightly increased in transformed cells, neither the CDK7 level nor the CDK7 and cyclin H localizations were altered when compared with those in normal CM. The results suggest the absence of the CDK inhibitor (CKI) p21 in two of the three melanoma cell lines and, as a main feature, a striking underexpression of p27 in the three transformed cell lines. Finally, although the p16 level was almost the same in normal and transformed cells, a loss of p16-CDK4 interaction was observed in two of the three melanoma cell lines. CONCLUSIONS: Deregulated expression of G1 cyclins and CKIs and alteration in the interaction of CKIs with CDKs may be implicated in the neoplastic transformation of human ocular melanocytes to malignant melanoma cells.

G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells.

We have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3-kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole-induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002-induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up-regulation of the CDK inhibitor p27Kip1 and to its increased association to CDK2.

Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates.

DNA replication is an asymmetric process involving concurrent DNA synthesis on leading and lagging strands. Leading strand synthesis proceeds concomitantly with fork opening, whereas synthesis of the lagging strand essentially takes place on a single-stranded template. The effect of this duality on DNA damage processing by the cellular replication machinery was tested using eukaryotic cell extracts and model DNA substrates containing site-specific DNA adducts formed by the anticancer drug cisplatin or by the carcinogen N-2-acetylaminofluorene. Bypass of both lesions was observed only with fork-like substrates, whereas complete inhibition of DNA synthesis occurred on damaged single-stranded DNA substrates. These results suggest a role for additional accessory factors that permit DNA polymerases to bypass lesions when present in fork-like DNA.

In vitro bypass replication of the cisplatin-d(GpG) lesion by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase is highly mutagenic.

Eukaryotic DNA polymerase beta and the reverse transcriptases are the most inaccurate of the known DNA polymerases. We report here mutagenic replication in vitro past intrastrand N(7)G-N(7)G chelates of the cis-diamminedichloroplatinum(II), the major DNA adduct of the antitumor agent cisplatin by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase (42% and 26% mutations, respectively). The most frequent modifications generated by both enzymes were one-base frameshift deletions. Only one mutational hot spot opposite the platinated guanines was observed with human immunodeficiency virus type I reverse transcriptase, while two hot spots were generated by DNA polymerase beta, one at the base situated 5' to the lesion and the other situated 4-6 nucleotides 5' to the adduct. An unusual mutagenic event, tandem replication of a 12-base pair sequence, was observed with DNA polymerase beta. The mutational spectra of the two DNA polymerases suggest that template slippage occurred with higher frequency in the presence of the more distributive DNA polymerase beta.

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.

Replication of DNA containing cisplatin lesions and its mutagenic consequences.

Cisplatin [cis-diamminedichloroplatinum II] is widely used in the treatment of a broad range of tumors. A number of biological and biochemical results indicate that the reaction of cisplatin with DNA is responsible for the cytotoxic action of the drug. However, cisplatin can induce mutagenesis and may be carcinogenic in humans. Error prone replication of damaged DNA must be considered as a possible mechanism of mutagenesis. In this short review, we present data indicating that DNA containing cisplatin lesions can be replicated by prokaryotes and eukaryotes in a mutagenic fashion.

Effect of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum (II) on the activity of a helicase essential for DNA replication, the herpes simplex virus type-1 origin-binding protein.

To determine the effect of the major DNA adduct, the intrastrand d(GpG) cross-link, produced by the antitumor drug cis-diamminedichloroplatinum(II) on the activity of a helicase known to be essential for DNA replication, we have examined its interaction with the origin-binding protein (UL9 protein) of herpes simplex virus type-1. We found that the helicase activity of the UL9 protein is inhibited only when the adduct is present on the template strand along which the protein translocates. This effect was paralleled by a comparable inhibition of the UL9 protein's DNA-dependent ATPase activity. The inhibitory effect of the lesion can be reduced by the addition of the herpes simplex virus type-1 single-stranded DNA-binding protein, ICP8. This stimulatory effect is specific for ICP8 and appears to be the result of the functional and physical interaction that is known to exist between the UL9 protein and ICP8, and not due to the preferential interaction of ICP8 with the adduct.

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(II) adduct placed on codon 13 of the human H-ras proto-oncogene.

Cisplatin (cis-[Pt(NH3)2Cl2]) is a widely used antitumor agent whose mutagenic activity raises the possibility of the induction of secondary cancer as a result of treatment. Mutation of the proto-oncogene H-ras is found in more than 30% of all human tumors, where it has been postulated to contribute to the initiation and progression of human cancers. Activating mutations in the H-ras gene are predominantly single-base substitutions, most frequently at codons 12, 13 and 61. In the present work we have studied the mutational spectra induced by a single cis-[Pt(NH3)2d(GpG)] adduct, the most frequent DNA crosslink formed by cisplatin. We have constructed a 25-mer-Pt oligonucleotide singly modified at codon 13 (GGT) within the human H-ras DNA sequence and we have inserted it into a single-stranded SV40-based shuttle vector able to replicate in simian COS7 cells. After replication in the mammalian host, vectors were extracted, amplified in bacteria and DNA from 124 randomly chosen colonies was sequenced. The observed mutation frequency was 21%. Base substitutions were the most frequent modification. 92% of the mutagenic events occurred at one or both of the platinated guanines of codon 13. The single G-->T transversion accounted for 65% of the total mutations scored. All single base substitutions were located at the G in the 3' position showing, for the first time, that the guanine at the 3' side of a cis-[Pt(NH3)2d(GpG)] adduct may be a preferential site for cisplatin induced mutations. The substitution G-->T at this position of the codon 13 of the H-ras proto-oncogene is known to induce the oncogenic properties of the p21ras protein.

Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA.

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.

Characterization and localization of cis-diamminedichloro-platinum(II) adducts on a purified oligonucleotide containing the codons 12 and 13 of H-ras proto-oncogene.

The use of substrates containing well defined adducts at precise sites, is required to perform a careful analysis of the toxic and mutagenic potential of a lesion. As a first step in this direction the octamer 5'-d(CCGGCGGT), containing the sequence of the codons 12 d(GGC) and 13 d(GGT) of the human H-ras gene, was reacted with the antitumoral drug cis-diamminedichloroplatinum(II). The platinated products have been purified by HPLC. A first set of experiments, including enzymatic digestions with nuclease P1 followed by alkaline phosphatase and acid-catalysed hydrolysis, allowed us to determine which bases were engaged in the cis-DDP lesions. Our results indicate that only guanine residues were chelated with cisplatin to yield bifunctional adducts. Furthermore, by performing enzymatic digestions with phosphodiesterases, we have located the adducts with respect to the 5' end of the octamer. Among the purified and characterized platinated oligonucleotides, three present a particular interest, since we have shown here that the cis-d(GpG) adduct is precisely situated either at the d(GGC) or at the d(GGT) or at both sites of their sequence.