Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.

Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.

[Regulatory effect of faciogenital dysplasia 6 gene on hepatic stem cell differentiation].

Objective: To investigate the regulatory effect of faciogenital dysplasia 6 (FGD6) gene on hepatic stem cell differentiation. Methods: FGD6 gene was selected for the co-intervention of target sequence, the AdEasy system was used for the construction of adenovirus vector and the packaging and multiplication of the recombinant adenovirus vector pSES-FGD6-siRNA, and the HP14.5 cells were infected. Immunofluorescence assay was used to measure the expression of FGD6 protein in HP14.5 cells, quantitative real-time PCR was used to measure the mRNA expression of FGD6, alpha-fetoprotein (AFP), and albumin (Alb), and Western blot was used to measure the protein expression of FGD6, AFP, and Alb. The empty pSES-Ad-RFP adenovirus vector was constructed as control in each group. All data were expressed as x±s, and a one-way analysis of variance was performed. Results: FGD6 protein was mainly expressed in the nucleus of HP14.5 cells. The pSES-FGD6-siRNA adenovirus vector was successfully constructed and it downregulated the expression of FGD6 gene and the mRNA and protein expression of AFP in HP14.5 cells and upregulated the mRNA and protein expression of Alb (P < 0.01). Conclusion: The inhibition of the expression of FGD6 gene in HP14.5 cells may differentiate HP14.5 cells into hepatocytes. Therefore, FGD6 gene plays an important role in the differentiation regulation of hepatic stem cells.

[Clinical observation of partial pancreatectomy as part of primary cytoreductive surgery in advanced epithelial ovarian cancer].

OBJECTIVE: The aim of this study is to evaluate the safety and efficacy of partial pancreatectomy as part of primary cytoreductive surgery in advanced epithelial ovarian cancer (EOC). METHODS: A total of 8 patients were recruited in this study who underwent partial pancreatectomy during the primary cytoreductive surgeries for advanced EOC in Fudan University Shanghai Cancer Center from April 2009 to July 2015. Their clinicopathological characteristics, diameter of metastatic tumors, the scope of cytoreductive surgeries, residual diseases after cytoreductive surgeries, postoperative complications and survival situation were retrospective analyzed. RESULTS: (1) Clinicopathological characteristics: the median age of these patients was 58 years old (range: 39-63 years old) . The median value of preoperative serum CA125 was 1 688 kU/L (range: 119-5 000 kU/L) . The median diameter of metastatic tumors involved in pancreatic body or tail was 4.5 cm (range: 3-10 cm). All the tumors from the 8 patients were confirmed to be high-grade serous carcinoma. Four patients were staged as International Federation of Gynecology and Obstetrics (FIGO) Ⅳ, and the other 4 patients were staged as FIGO Ⅲc. (2) Tumor metastases and the scope of cytoreductive surgeries: all of these 8 patients had widely disseminated ovarian cancer, with involvement of upper abdominal, middle abdominal and pelvic cavity. Each patient underwent extensive intra-abdominal cytoreductive surgeries, including hysterectomy, bilateral salpingo-oophorectomy, omentectomy, pelvic peritonectomy, splenectomy, partial pancreatectomy. Each patient had cytoreductive surgeries of 9.6 different sites on average. Of all 8 patients who underwent partial pancreatectomy, 7 patients had pancreatic tails removed; the other 1 patient had pancreatic body and tail removed. The median volume of blood loss during surgery was 1 350 ml (range: 300-3 500 ml) , blood transfusion was performed in 7 patients with the median volume of 1 150 ml (range: 500-1 800 ml). (3) Residual diseases after cytoreductive surgeries: optimal cytoreduction was achieved in all patients, with microscopic residual disease in 3 patients, residual tumors diameter < 0.5 cm in 3 patients, and residual tumors diameter between 0.5 and 1 cm in 2 patients. (4) Postoperative complications: 4 patients suffered from complications including pancreatic leakage (2/8), intraperitoneal hemorrhage (1/8) and pancreatic pseudocyst accompanied by infection (1/8). These complications were treated successfully by conservative managements. (5) Survival situation: during the median follow-up duration of 17 months (ranged from 2 to 46 months), 5 patients were still alive until the end of follow-up, including 4 cases under treatment and 1 case survived 29 months without relapse after treatment. Three patients were respectively died in 5, 20 and 46 months after surgery. CONCLUSION: There is a higher risk of postoperative complications of pancreas resection as part of primary cytoreductive surgery in advanced epithelial ovarian cancer, but the resection of pancreatic metastases and part of the pancreas is feasible and necessary.

Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells.

Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

Overexpression of Wnt-2 in colorectal cancers.

UNLABELLED: The binding of the Wnt ligand to its receptor Frizzled, activates the Wnt canonical signaling pathway in carcinogenesis as well as many cellular processes, including cellular proliferation and differentiation. Wnt-2, one of 19 members of the Wnt gene family, is frequently overexpressed in malignant tissues. Here, in order to investigate the role of Wnt-2 in colorectal carcinogenesis, we examined the expression of the Wnt-2 protein in 120 colorectal cancers by immunohistochemistry. Wnt-2 protein was expressed in the cell membrane and cytoplasm and up-regulated in 74 (61.7%) of 120 colorectal cancers. Statistically, overexpression of Wnt-2 protein was not associated with the clinical and pathological parameters studied, including tumor location, tumor size, clinical stage, lymph node metastasis, and 5-year survival (P > 0.05). These results indicate that up-regulation of the Wnt-2 protein might play a role in the development of colorectal cancers, as an early event of carcinogenesis. KEYWORDS: Wnt-2 protein, expression, immunohistochemistry, tissue microarray, colon cancer.

Correction to: Role of intestinal microbiome in American ginseng-mediated colon cancer prevention in high fat diet-fed AOM/DSS mice.

This article was originally published with the wrong title.

Role of intestinal microbiome in American ginseng-mediated colon cancer prevention in high fat diet-fed AOM/DSS mice [corrected].

OBJECTIVE: Chronic intestinal inflammation is a risk factor for colorectal cancer (CRC) initiation and development. Diets that are rich in Western style fats have been shown to promote CRC. This study was conducted to investigate the role of intestinal microbiome in American ginseng-mediated CRC chemoprevention in a mouse model. The population and diversity of enteric microbiome were evaluated after the ginseng treatment. METHODS: Using an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced gut inflammation and tumorigenesis mouse model, the effects of oral American ginseng on high fat diet-associated enteric pathology were determined. After establishment of a 16S rRNA illumina library from fecal samples, MiSeq sequencing was carried out to reveal the microbial population. The alpha and beta diversities of microbiome were analyzed. RESULTS: American ginseng significantly attenuated AOM/DSS-induced colon inflammation and tumorigenesis by reducing the colitis score and colon tumor multiplicity. The MiSeq results showed that the majority of sequences fell into three phyla: Firmicutes, Bacteroidetes and Verrucomicrobia. Further, two significant abundance shifts at the family level, Bacteroidaceae and Porphyromonadaceae, were identified to support ginseng's anti-colitis and anti-tumor effects. In addition, alpha and beta diversity data demonstrated that ginseng led to a profound recovery from the AOM/DSS-induced dysbiosis in the microbial community. CONCLUSION: Our results suggest that the CRC chemopreventive effects of American ginseng are mediated through enteric microbiome population-shift recovery and dysbiosis restoration. Ginseng's regulation of the microbiome balance contributes to the maintenance of enteric homeostasis.

The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression.

Inactivation of the adenomatous polyposis coli (APC) gene is a critical event in the development of human colorectal cancers. At the biochemical level, several functions have been assigned to the multidomain APC protein, but the cellular effects of APC expression and how they relate to its biochemical functions are less well defined. To address these issues, we generated a recombinant adenovirus (Ad-CBR) that constitutively expresses the central third of APC, which includes all of the known beta-catenin binding repeats. When expressed in colon cancer cells, Ad-CBR blocked the nuclear translocation of beta-catenin and inhibited beta-catenin/Tcf-4-mediated transactivation. Accordingly, expression of endogenous targets of the APC/beta-catenin/Tcf-4 pathway was down-regulated. Ad-CBR infection of colorectal cancer cell lines with mutant APC but wild-type beta-catenin resulted in substantial growth arrest followed by apoptosis. These effects were attenuated in lines with wild-type APC but with mutated beta-catenin. These findings suggest that the beta-catenin-binding domain in the central third of APC is sufficient for its tumor suppressor activity.

CDX2 is mutated in a colorectal cancer with normal APC/beta-catenin signaling.

The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

The future of flexor tendon surgery.

Clinical outcomes following flexor tendon repair have made significant improvements in the last 50 years. In that time standard treatment has evolved from secondary grafting to primary repair with postoperative rehabilitation protocols. Unfortunately, excellent results are not yet attained universally following treatment. Improving understanding of tendon healing at the cellular, molecular, and genetic levels will likely enable surgeons to modulate the normal repair process. We now look toward biologic augmentation of flexor tendon repairs to address the problems of increasing tensile strength while reducing adhesion formation following injury and operative repair.

Adenoviral vector-mediated gene transfer for human gene therapy.

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.

Fluorescence-based functional assay for Wnt/beta-catenin signaling activity.

Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, beta-catenin forms a complex with the lymphocyte enhancer-binding factor/T cell factor transcription factor and activates downstream targets that promote cell proliferation. Here we developed a Wnt-dependent beta-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length beta-catenin, and a GAL4-responsive reporter expressing GFP. The chimeric transcription factor was highly unstable and exerted no detectable transactivating effect on the GAL4-responsive reporter. However, lithium and Wnt1 significantly stabilized this chimeric transactivator, indicating that this transactivation system is regulated by beta-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate beta-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit beta-catenin signaling in human tumors.

Induced maturation of hepatic progenitor cells in vitro.

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

The antitumor natural compound falcarindiol promotes cancer cell death by inducing endoplasmic reticulum stress.

Falcarindiol (FAD) is a natural polyyne with various beneficial biological activities. We show here that FAD preferentially kills colon cancer cells but not normal colon epithelial cells. Furthermore, FAD inhibits tumor growth in a xenograft tumor model and exhibits strong synergistic killing of cancer cells with 5-fluorouracil, an approved cancer chemotherapeutic drug. We demonstrate that FAD-induced cell death is mediated by induction of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Decreasing the level of ER stress, either by overexpressing the ER chaperone protein glucose-regulated protein 78 (GRP78) or by knockout of components of the UPR pathway, reduces FAD-induced apoptosis. In contrast, increasing the level of ER stress by knocking down GRP78 potentiates FAD-induced apoptosis. Finally, FAD-induced ER stress and apoptosis is correlated with the accumulation of ubiquitinated proteins, suggesting that FAD functions at least in part by interfering with proteasome function, leading to the accumulation of unfolded protein and induction of ER stress. Consistent with this, inhibition of protein synthesis by cycloheximide significantly decreases the accumulation of ubiquitinated proteins and blocks FAD-induced ER stress and cell death. Taken together, our study shows that FAD is a potential new anticancer agent that exerts its activity through inducing ER stress and apoptosis.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.