Repeated intratumoral administration of ONCOS-102 leads to systemic antitumor CD8+ T-cell response and robust cellular and transcriptional immune activation at tumor site in a patient with ovarian cancer.

Adenoviruses are excellent immunotherapeutic agents with a unique ability to prime and boost immune responses. Recombinant adenoviruses cause immunogenic cancer cell death and subsequent release of tumor antigens for antigen presenting cells, resulting in the priming of potent tumor-specific immunity. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We report a case of a 38-year-old female with Stage 3 metastatic micropapillary serous carcinoma of the ovary. She was treated in a Phase I study with a granulocyte-macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in progressive infiltration of CD8+ lymphocytes into the tumor and concomitant systemic induction of several tumor-specific CD8+ T-cell populations. The patient was alive at the latest follow up more than 20 months after initiation of the study.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation.

BACKGROUND: Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored. METHODS: Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit. RESULTS: We show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors. CONCLUSION: Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.

p21 does not protect cancer cells from apoptosis induced by nongenotoxic p53 activation.

p21(Waf1/Cip1) is a p53 transcription target implicated in both major functions of the tumor suppressor--cell cycle arrest and apoptosis. It is a potent inhibitor of the key cyclin-dependent kinases (CDK1-4), and has been thought to be the main mediator of p53-dependent G1 and G2 arrest. However, an increasing body of information suggests that in addition to its cell-cycle inhibitory activity, p21 can affect p53-dependent apoptosis. These data have been obtained from experiments in which p53 is activated primarily by genotoxic stress. In this study, we use the selective MDM2 antagonist, nutlin-3a, as a nongenotoxic p53 activator and show that the cell-cycle arrest function of p21 is dependent on the cellular context. In most cancer cell lines, p53-dependent p21 induction is essential for cell-cycle arrest, but in some, p21 is dispensable. Depletion of p21 did not increase the apoptotic response to nutlin-3a in all seven cancer cell lines tested and p21 overexpression did not protect apoptosis-sensitive lines from death. p21 was found to mediate nutlin-induced p53-dependent downregulation of another antiapoptotic protein, survivin, without significantly affecting the apoptotic outcome. Taken together our results suggest that p21 induction does not affect the apoptotic response to nongenotoxic p53 activation.

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells.

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.

Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1.

MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Recently, small-molecule antagonists of MDM2, the Nutlins, have been developed to inhibit the p53-MDM2 interaction and activate p53 signaling. However, half of human cancers have mutated p53 and they are resistant to Nutlin treatment. Here, we report that treatment of the p53-mutant malignant peripheral nerve sheath (MPNST) and p53-null HCT116 cells with cisplatin (Cis) and Nutlin-3a induced a degree of apoptosis that was significantly greater than either drug alone. Nutlin-3a also increased the cytotoxicity of both carboplatin and doxorubicin in a series of p53-mutant human tumor cell lines. In the human dedifferentiated liposarcoma cell line (LS141) and the p53 wild-type HCT116 cells, Nutlin-3a induced downregulation of E2F1 and this effect appeared to be proteasome dependent. In contrast, in MPNST and HCTp53-/- cells, Nutlin-3a inhibited the binding of E2F1 to MDM2 and induced transcriptional activation of free E2F1 in the presence of Cis-induced DNA damage. Downregulation of E2F1 by small interfering RNA significantly decreased the level of apoptosis induced by Cis and Nutlin-3a treatment. Moreover, expression of a dominant-negative form of E2F1 rescued cells from apoptosis, whereas cells overexpressing wild-type E2F1 showed an increase in cell death. This correlated with the induction of the proapoptotic proteins p73alpha and Noxa, which are both regulated by E2F1. These results indicate that antagonism of MDM2 by Nutlin-3a in cells with mutant p53 enhances chemosensitivity in an E2F1-dependent manner. Nutlin-3a therefore may provide a therapeutic benefit in tumors with mutant p53 provided it is combined with chemotherapy.

Targeting the p53-MDM2 interaction to treat cancer.

The tumour suppressor p53 is a transcription factor with powerful antitumour activity that is controlled by its negative regulator MDM2 (mouse double minute 2, also termed HDM2 in humans) through a feedback mechanism. MDM2, which is overproduced in many tumours, binds p53 and inhibits its function by modulating its transcriptional activity and stability. Activation of p53 in tumour cells by inhibiting its physical interaction with MDM2 has been in the focus of cancer drug discovery. However, development of nonpeptidic MDM2 antagonists turned out to be challenging. Recently, the first potent and selective small-molecule antagonists of MDM2, the Nutlins, have been identified. Studies with Nutlins provided in vitro and in vivo proof-of-principle for targeting p53-MDM2 interaction for cancer therapy.

Cell-based screening approach for antitumor drug leads which exploits sensitivity differences between normal and cancer cells: identification of two novel cell-cycle inhibitors.

A cell-based in vitro screening approach for identification of antitumor drug leads that exploits the differential sensitivity between normal and cancer cells was developed. It is a three-step, high-throughput screen for antiproliferative and/or cytotoxic activity measured by a 7 day MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromidel assay using small panels of proliferating primary human cells and established cancer cell lines. Proof-of-concept experiments successfully identified 11 known cancer drugs randomly mixed with 5000 test compounds. Application of this screening approach to a library of 110000 compounds allowed for the identification of several novel chemical classes of compounds active against an expanded panel of cancer cell lines in vitro. Two of the compounds representing novel mitotic inhibitors with in vivo potency against established breast cancer xenografts (MDA-MB-435) are reported here.

Quantification of telomerase activity by direct scintillation counting.

An improved telomerase assay was developed that allows direct quantification of the enzyme activity by scintillation counting of the labeled telomerase product. The assay measures the incorporation of 32P-dGTP into telomeric repeats synthesized at the 3' end of a biotinylated primer. Telomerase reaction product is separated from the reaction mix by streptavidin-coated magnetic beads and counted. The assay can be used for quantitative studies of human telomerase and its inhibitors.

Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.

By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A+T rich and contained different regulatory elements. By using a two step procedure (Heparin-sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD.

Guide to identification of origins of DNA replication in eukaryotic cell chromosomes.

Several experimental approaches for identification of origins of DNA replication have been developed recently that allow, for the first time, unique initiation sites in mammalian chromosomes to be mapped at single-copy loci. A brief description of the rationale, advantages, and limitations has been provided for each approach, as well as information that can help the reader choose the method(s) most suitable for a particular system. The various methods are divided into three groups: (1) analysis of nascent DNA strands, (2) analysis of DNA structures, and (3) analysis of origin activity (i.e., ability to support autonomous replication). It is hoped that this information will serve as a practical guide for identifying new origins of replication.

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells.

Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.

Identification of an origin of bidirectional DNA replication in mammalian chromosomes.

Mechanistically, an origin of bidirectional DNA replication (OBR) can be defined by the transition from discontinuous to continuous DNA synthesis that must occur on each template strand at the site where replication forks originate. This results from synthesis of Okazaki fragments predominantly on the retrograde arms of forks. We have identified these transitions at a specific site within a 0.45 kb sequence approximately 17 kb downstream from the 3' end of the dihydrofolate reductase gene in Chinese hamster ovary chromosomes. At least 80% of the replication forks in a 27 kb region emanated from this OBR. Thus, initiation of DNA replication in mammalian chromosomes uses the same replication fork mechanism previously described in a variety of prokaryotic and eukaryotic genomes, suggesting that mammalian chromosomes also utilize specific cis-acting sequences as origins of DNA replication.

Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments.

We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells.

Kinetics of replicon initiation during S phase of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [3H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.