Patrick W.K. Lee

The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus

NIH‐3T3 cells, which are resistant to reovirus infection, became susceptible when transformed with activated Sos or Ras. Restriction of reovirus proliferation in untransformed NIH‐3T3 cells was not at the level of viral gene transcription, but rather at the level of viral protein synthesis. An analysis of cell lysates revealed that a 65 kDa protein was phosphorylated in untransformed NIH‐3T3 cells, but only after infection with reovirus. This protein was not phosphorylated in infected or uninfected transformed cells. The 65 kDa protein was determined to be the double‐stranded RNA‐activated protein kinase (PKR), whose phosphorylation leads to translation inhibition. Inhibition of PKR phosphorylation by 2‐aminopurine, or deletion of the Pkr gene, led to drastic enhancement of reovirus protein synthesis in untransformed cells. The emerging picture is one in which early viral transcripts trigger PKR phosphorylation in untransformed cells, which in turn leads to inhibition of translation of viral genes; this phosphorylation event is blocked by an element(s) in the Ras pathway in the transformed cells, allowing viral protein synthesis to ensue. The usurpation of the Ras signaling pathway therefore constitutes the basis of reovirus oncolysis.

DNA-dependent protein kinase acts upstream of p53 in response to DNA damage.

The tumour suppressor p53 becomes activated as a transcription factor in response to DNA damage, but the mechanism for this activation is unclear. A good candidate for an upstream activator of p53 is the DNA-dependent protein kinase (DNA-PK) that depends on the presence of DNA breaks for its activity. Here we investigate the link between DNA damage and the activation of DNA-PK and of p53. To determine whether DNA-PK is an upstream mediator of the p53 DNA-damage response, we analysed a severe combined-immunodeficiency (SCID) mouse cell line, SCGR11 (refs 7, 8), and the human glioma cell line M059J (ref. 9). Both cell lines lack any detectable DNA-PK activity. We find that p53 is incapable of binding to DNA in the absence of DNA-PK, that DNA-PK is necessary but not sufficient for activation of p53 sequence-specific DNA binding, and that this activation occurs in response to DNA damage. Our results establish DNA-PK as a link between DNA damage and p53 activation, and reveal the existence of a mammalian DNA-damage-response pathway.

Aldehyde Dehydrogenase Activity of Breast Cancer Stem Cells Is Primarily Due To Isoform ALDH1A3 and Its Expression Is Predictive of Metastasis

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer. STEM CELLS 2011;29:32–45

Aldehyde dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1, one of 19 ALDH isoforms expressed in humans, was generally believed to be responsible for the ALDH activity of CSCs. More recently, experiments with murine hematopoietic stem cells, murine progenitor pancreatic cells, and human breast CSCs indicate that other ALDH isoforms, particularly ALDH1A3, significantly contribute to aldefluor positivity, which may be tissue and cancer specific. Therefore, potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators.

How p53 binds DNA as a tetramer

The p53 tumor suppressor protein is a tetramer that binds sequence‐specifically to a DNA consensus sequence consisting of two consecutive half‐sites, with each half‐site being formed by two head‐to‐head quarter‐sites (→← →←). Each p53 subunit binds to one quarter‐site, resulting in all four DNA quarter‐sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA‐binding domains of each dimer bound to either consecutive or alternating quarter‐sites. We show here that the two monomers within a dimer bind to a half‐site (two consecutive quarter‐sites), but not to separated (alternating) quarter‐sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half‐sites. Although one dimer within the tetramer is sufficient for binding to one half‐site in DNA, concurrent interaction of the second dimer with a second half‐site in DNA drastically enhances binding affinity (at least 50‐fold). This cooperative dimer–dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.

Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR−/− (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

Reovirus Oncolysis of Human Breast Cancer

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2−/− mouse embryo fibroblasts (MEFs), unlike Atm−/− or p53−/− MEFs, behaved like normal MEFs in manifesting p21 induction and G1 arrest upon exposure to γ-irradiation. Therefore, Chk2 is not involved in p53-mediated G1 arrest. To examine the role of Chk2 in p53-dependent apoptotic response, we used adenovirus E1A-expressing MEFs. We show that Chk2−/− cells, like p53−/− cells, did not undergo DNA damage-induced apoptosis, whereas Atm−/− cells behaved like normal cells in invoking an apoptotic response. Furthermore, this apoptosis could occur in the absence of protein synthesis, suggesting that it is preexisting, or “latent,” p53 that mediates this response. We conclude that Chk2 is not involved in Atm- and p53-dependent G1 arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis.

The α-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus

Abstract A series of synthetic sialosides were evaluated for their ability to interact with reovirus serotype 3. It was found that siolosides with terminal N-acetylneuraminic acid (NeuNAc) linked in either an α2,3 or α2,6 configuration effectively blocked the binding of reovirus to mouse L fibroblasts, in contrast to a monosaccharide mixture containing the oligosaccharide constituents. Direct binding of reovirus to the sialosides was also demonstrable using sialosides conjugated to bovine serum albumin as ligands in a solid phase binding system. Of particular significance was the finding that the conjugate containing α-sialic acid alone (linked to bovine serum albumin) was capable of being recognized by reovirus at a level comparable to that of the other sialoside conjugates. Virus binding was abrogated by pretreating such conjugates with neuraminidase. These results suggest that the α-anomeric form of sialic acid is the minimal receptor determinant for reovirus recognition.

DNA damage-induced apoptosis requires the DNA-dependent protein kinase, and is mediated by the latent population of p53

Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following γ‐irradiation, latent p53 formed a complex with the catalytic subunit of the DNA‐dependent protein kinase (DNA‐PKCS). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks. This association was accompanied by phosphorylation of pre‐existing, latent p53 at Ser18 (corresponding to Ser15 in human p53), which was not found in DNA‐PKCS−/− cells. Most significantly, DNA damage‐induced apoptosis was abolished in both DNA‐PKCS−/− and p53−/− cells. In addition, blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient for executing the apoptotic program. Finally, E1A‐expressing MEFs from a p53 ‘knock‐in’ mouse where Ser18 was mutated to an alanine had an attenuated apoptotic response, indicating that phosphorylation of this site by DNA‐PK is a contributing factor for apoptosis.