Xinjian Liu

Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy

In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2 (PGE2), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death–induced tumor repopulation pathway in which caspase 3 has a major role.

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinsons disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

Apoptotic Caspases Regulate Induction of iPSCs from Human Fibroblasts

The molecular mechanisms involved in the derivation of induced pluripotent stem cells (iPSCs) from differentiated cells are poorly understood. Here we report that caspases 3 and 8, two proteases associated with apoptotic cell death, play critical roles in induction of iPSCs from human fibroblasts. Activation of caspases 3 and 8 occurs soon after transduction of iPSC-inducing transcription factors. Oct-4, a key iPSC transcription factor, is responsible for the activation. Inhibition of caspase 3 or 8 in human fibroblast cells partially or completely (respectively) prevents the induction of iPSCs. Furthermore, retinoblastoma susceptibility (Rb) protein appears to be one of the factors that act downstream of the caspases. We propose that caspases are key facilitators of nuclear reprogramming in iPSC induction.

Caspase 3 Promotes Surviving Melanoma Tumor Cell Growth after Cytotoxic Therapy

Metastatic melanoma often relapses despite cytotoxic treatment, so the understanding of melanoma tumor repopulation is crucial to improving our current therapies. In this study, we aim to define the role of caspase 3 in melanoma tumor growth after cytotoxic therapy. We examined a paradigm-changing hypothesis that dying melanoma cells undergoing apoptosis during cytotoxic treatment activate paracrine signaling events that promote the growth of surviving tumor cells. We propose that caspase 3 plays a key role in the initiation of the release of signals from dying cells to stimulate melanoma tumor growth. We created a model for tumor cell repopulation in which a small number of luciferase-labeled, untreated melanoma cells are seeded onto a layer of a larger number of unlabeled, lethally treated melanoma cells. We found that dying melanoma cells significantly stimulate the growth of living melanoma cells in vitro and in vivo. Furthermore, we observed that caspase 3 gene knockdown attenuated the growth-stimulating effect of irradiated, dying cells on living melanoma cell growth. Finally, we showed that caspase 3-mediated dying melanoma cell stimulation of living cell growth involves secreted PGE2. Our study therefore suggests a counterintuitive strategy to inhibit caspase 3 for therapeutic gain in melanoma treatment.

Quantitative, Noninvasive Imaging of Radiation-Induced DNA Double-Strand Breaks In Vivo

DNA double-strand breaks (DSB) are a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA DSBs is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here, we report a novel approach to image and quantify DSBs in live mammalian cells through bifragment luciferase reconstitution. N- and C-terminal fragments of firefly luciferase genes were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DSBs, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C-luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, noninvasive quantification of DSBs in cells irradiated with X-rays and (56)Fe ions. Furthermore, it allowed for the evaluation of DSBs noninvasively in vivo in irradiated tumors over 2 weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase-based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DSB repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time.

Oncolytic adenovirus delivering herpes simplex virus thymidine kinase suicide gene reduces the growth of human retinoblastoma in an in vivo mouse model

Oncolytic conditionally replicating adenoviruses (CRAd) can exclusively replicate in and lyse tumor cells and are therefore promising tools in cancer therapy. In this study, we combined the oncolytic potential of a CRAd with its ability to deliver a suicide gene (herpes simplex virus thymidine kinase suicide gene, HSVtk) in order to further enhance tumor cell killing in a human retinoblastoma (RB) mouse model. We could demonstrate that CRAd driven by the human telomerase reverse transcriptase (hTERT) promoter and armed with the HSV thymidine kinase suicide gene/ganciclovir (HSVtk/GCV) could very effectively reduce growth of human RB in an orthotopic nude mouse model. These findings suggest that hTERT promoter-driven CRAd in combination with HSVtk/GCV gene therapy could be a promising new approach for the treatment of RB. In addition, we found that hTERT promoter-driven CRAd replication occurred exclusively in human RB cells but not in primary human retinal pigment epithelial cells (hRPE), indicating that application of hTERT promoter-driven CRAd for the treatment of RB would be safe.

Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression.

Dopaminergic (DA) neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson’s disease. A significant obstacle is the low efficiency of conversion during the reprogramming process. Here, we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons. In particular, blocking p53 activity using a dominant-negative p53 (p53-DN) in IMR90 cells increases the conversion efficiency by 5–20 fold. The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression, significant dopamine uptake and production capacities, and enables symptomatic relief in a rat Parkinson’s disease model. Taken together, our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons.

Novel strategies to augment genetically delivered immunotoxin molecular therapy for cancer therapy

Immunotoxin therapy is a promising molecular cancer treatment strategy. Its main advantage is seletive cytotoxicity towards tumor cells and minimal toxicity in normal tissues. However, a short half-life and rapid clearance severely hampers its clinical application. We report here a novel genetic approach in which a recombinant adenovirus vector was used to deliver an immunotoxin gene e23(scFv)-PE40 targeted to the oncogene c-erbB-2 (also known as Her2/neu). This vector, when combined with a low dose of a conditionally replicative adenovirus vector (CRAd), has enhanced tumor-killing ability either alone or in combination with the chemotherapeutic agent etoposide. Our data show that low-dose CRAd facilitated the replication of replication-deficient Ad-e23(scFv)-PE40 up to 6–20 times and the transcription of e23(scFv)-PE40 gene up to 12 times. Moreover, etoposide increased the e23(scFv)-PE40 transcription up to 8.5 times. Furthermore, we show that systemic application of Ad-e23(scFv)-PE40 and enhanced expression of the immunotoxin gene was well tolerated as determined by serum biochemical markers and histological examination of most vital organs. Taken together, our data support a novel genetic immunotoxin delivery approach that may yield enhanced efficacy against a variety of Her2/neu-expressing tumors.

Enhanced transduction and improved photoreceptor survival of retinal degeneration by the combinatorial use of rAAV2 with a lower dose of adenovirus

Recombinant adeno-associated virus (rAAV) is widely used in retinal gene therapy. Enhanced rAAV transduction may be important for better therapeutic effects in some retinal gene therapies. In this study, we examined the effects of adenovirus 5 (Ad5) on retina transduction mediated by rAAV2. Our results provide the first evidence that low levels of either replication-incompetent or conditional replication-competent Ad5 significantly enhance and accelerate transgene expression in human and rat retinal cells. This effect occurs principally at the transcriptional level, rather than through enhanced viral entry or DNA replication. In in vivo analyses with the SD rat, the Balb/c mouse, and the RCS rat, strong enhancement and acceleration of transgene expression, as well as therapeutic effects, were confirmed. Low levels of Ad5 may enhance the utility of rAAV2-mediated transduction strategies in future clinical investigations.

Distinctive Gene Transduction Efficiencies of Commonly Used Viral Vectors in the Retina

The transduction efficiency and cell tropism of viral vectors rAAV2/1, rAAV2, Ad5, Ad5/F35, and Lentivirus were evaluated in retina. All viral vectors achieved efficient transduction in living rat retina. However, each vector showed distinctive efficiency in vitro especially for rAAV2/1, which displayed poor transduction in cultured retinal cells. Distinctive cell-specific GFP expression was observed in vivo and in vitro for the same viral vector. The cell-specific tropism was not strictly correlated with the correspondent distribution of viral receptors in retina. These results provided important insights into the selection of appropriate vectors when specific retinal diseases are considered for gene therapy.