Biomedical subjects

Bingliang Fang

Publications and source records attributed to Bingliang Fang.

48 records · Page 3Linked to original sources

PubMed2002

A novel single tetracycline-regulative adenoviral vector for tumor-specific Bax gene expression and cell killing in vitro and in vivo.

Using a binary adenoviral system, we recently showed that the human telomerase reverse transcriptase (hTERT) promoter induces tumor-specific Bax gene expression. However, the strong cytotoxicity of Bax and other pro-apoptotic genes to packaging 293 cells has so far hindered construction of the desired single adenoviral vectors expressing toxic genes. We report here the construction of a single bicistronic adenoviral vector for tumor-specific Bax expression. The vector (Ad/gBax) utilizes the Tet-Off system and expresses a GFP/Bax fusion protein for easy detection. The hTERT promoter drives the expression of tTA, a transactivator capable of binding to TRE (tetracycline-responsive element) in the absence of tetracycline, which in turn induces expression of the GFP-Bax gene. The addition of tetracycline in 293 cells blocks the binding of tTA to TRE and substantially inhibits GFP-Bax expression and toxicity, thus allowing the packaging and production of Ad/gBax. Our data show that Ad/gBax could drive the high expression of GFP-Bax in tumor cells but not in normal cells and mouse tissues. Furthermore, the expression of GFP-Bax fusion protein elicited tumor-specific apoptosis in a variety of human cancer cells in vitro and in vivo at a level comparable to that induced by the binary system. Thus, Ad/gBax may become a potent therapeutic agent for the treatment of cancers.

Adenoviridae↗

PubMed2002

Targeted expression of green fluorescent protein/tumor necrosis factor-related apoptosis-inducing ligand fusion protein from human telomerase reverse transcriptase promoter elicits antitumor activity without toxic effects on primary human hepatocytes.

Liver toxicity is the major concern for use of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proteins in treatment of cancers. Here we report that normal human primary hepatocytes (NHPHs) are susceptible to the transduction of the wild-type, full-length coding sequence of the human TRAIL gene. To minimize potential toxicity of the TRAIL gene, a bicistronic adenoviral vector that expresses the green fluorescent protein/TRAIL fusion protein from the human telomerase reverse transcriptase promoter (designated Ad/gTRAIL) was constructed. In vitro and in vivo studies have showed that treatment with the adenoviral vector Ad/gTRAIL results in high-level expression of green fluorescent protein/TRAIL in cancer cells but no detectable transgene expression in NHPHs or in normal mouse liver tissues. Furthermore, treatment with Ad/gTRAIL effectively elicited apoptosis in malignant cells but not in NHPHs in vitro and suppressed tumor growth and prolonged duration of survival in vivo. Thus, with the combined advantages of the TRAIL gene and the human telomerase reverse transcriptase target, Ad/gTRAIL can be a potent therapeutic agent for the treatment of cancers.

Adenoviridae↗

PubMed2002

Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo.

A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.

Adenoviridae↗

PubMed2002

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis.

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.

Apoptosis↗

PubMed2002

Suppression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences.

Our previous studies showed that the urokinase-type plasminogen activator receptor (uPAR) and the p16 tumor suppressor gene play a significant role in glioma invasion. We expected that downregulation of uPAR and overexpression of p16 using a bicistronic vector might cause a additive and cooperative effect in the suppression of glioma invasion and growth. The bicistronic construct (Ad-uPAR/p16)-infected glioblastoma cell lines had significantly lower levels of uPAR and higher levels of p16 than controls. Cell cycle analysis showed the bicistronic vector caused G0/G1 arrest of the cell cycle. In vitro glioblastoma cell growth and invasiveness were inhibited in Ad-uPAR/p16-infected cells compared with controls. Ad-uPAR/p16 suppressed the tumor growth of glioblastoma cell lines in an ex vivo intracerebral tumor model and an in vivo subcutaneous tumor model. Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Adenoviridae↗

PubMed2002

Bax-induction gene therapy of pancreatic cancer.

BACKGROUND: Bax is a strong pro-apoptotic gene that induces programmed cell death when expressed. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit for telomerase, an enzyme found to be active in more than 85% of human cancers. Recently, a binary adenoviral system (Ad/GT-Bax + Ad/hTERT-GV16) was constructed using the hTERT promoter to induce Bax gene expression in tumor cells. METHODS: To test whether human pancreatic tumor cells would respond to this system of Bax-induced apoptosis, we compared the effects of Bax gene induction with that of LacZ gene induction using the same binary system. RESULTS: Lysates of the human pancreatic cell lines PANC-28, MIA PaCa-2, and BxPC-3 showed significantly elevated levels of human telomerase using the PCR-based TRAP assay. As early as 24 h after treatment with Bax-induction gene therapy, growth inhibition was observed. Overexpression of the Bax protein was confirmed by Western blotting. Extensive apoptosis on FACS analysis at 48 h was seen after Bax induction. In addition, cytosolic cytochrome c levels increased compared to mitochondrial levels after Bax induction. Levels of caspase-3, a key downstream enzyme involved in apoptosis, also increased significantly compared to controls after treatment. None of these effects were seen with LacZ. CONCLUSION: Our results suggest that the binary adenoviral vector system, Ad/GT-Bax + Ad/hTERT-GV16, induces high levels of Bax expression that induce apoptosis in human pancreatic cancer cells.

Apoptosis↗

PubMed2002

[Action of apoptosis-induced ligand gene in relation to tumor necrosis factor on human colon cancer cell line HT29].

OBJECTIVE: To evaluate the gene therapeutic efficiency of apoptosis-inducing ligand (TRAIL) related to tumor necrosis factor on human colon cancer cell line HT29. METHODS: Human colon cancer cell line HT29 was transfected with adenovirus-mediated TRAIL gene Ad/GT-TRAIL. The morphological changes, cell growth and apoptosis were measured by phase contrast microscope, MTT method and flow cytometry. RESULTS: Obvious morphological changes in HT29 cells was induced by Ad/GT-TRAIL and Ad/PGK-GV16. The cell suppression percentage and the percentage of apoptotic cells were 54.3% and 11.1%, respectively. When used in combination with Ad/PGK-GV16, HT29 was suppressed to 82.7% and the percentage of apoptotic cells was 24.6%. This result showed significantly enhanced therapeutic efficiency on HT29 and thus inhibiting of its growth (P < 0.05). CONCLUSION: Ad/GT-TRAIL is able to induce apoptosis of HT29 and inhibit its growth. Ad/GT-TRAIL shows significantly enhanced therapeutic efficiency for HT29 when used in combination with Ad/PGK-GV16.

Adenoviridae↗

PubMed2001

Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.

Alterations in intracellular Ca(2+) homeostasis and cytochrome c release from mitochondria have been implicated in the regulation of apoptosis, but the relationship between these events remains unclear. Here we report that enforced expression of either Bax or Bak via adenoviral gene delivery results in the accumulation of the proteins in the endoplasmic reticulum (ER) and mitochondria, resulting in early caspase-independent BCL-2-sensitive release of the ER Ca(2+) pool and subsequent Ca(2+) accumulation in mitochondria. The inhibition of ER-to-mitochondrial Ca(2+) transport with a specific inhibitor of mitochondrial Ca(2+) uptake attenuates cytochrome c release and downstream biochemical events associated with apoptosis. Bax and Bak also directly sensitize mitochondria to cytochrome c release induced by immediate emptying of ER Ca(2+) pool. Our results demonstrate that the effects of the "multidomain" proapoptotic BCL-2 family members Bak and Bax involve direct effects on the endoplasmic reticular Ca(2+) pool with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release. These effects modulate the kinetics of cytochrome c release and apoptosis.

Apoptosis↗

PubMedDate not supplied

A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity.

Using a binary co-transfection strategy of Ad/GT Bax and Ad/PGK-GV16, we have succeeded in inducing overexpression of Bax protein in three prostate cell lines (androgen-insensitive DU145 and PC3, and androgen-sensitive LNCaP). The expression of Bax protein by this system was sufficient to induce all three prostate lines to undergo apoptosis. The fact that DU145 cells which have a p53 mutation and are deficient in Bax, responded to this treatment, suggests that this effect is independent of these pathways. Initiation of the cleavage of Caspase-3 (CPP32/Yama/apopain) and PARP (poly (ADP-ribose) polymerase) by the introduction of Bax were confirmed by western blot analysis. Bcl-2 expression is relevant in the progression of prostate cancer and contributes to an androgen, apoptotic-resistant phenotype in the advanced stages. We examined stable Bcl-2 overexpressing DU145, PC3 and LNCaP cell lines as models of advanced prostate cancer. The adenoviral co-transfection system induced Bax protein expression and apoptosis even in these Bcl-2 transfected cell lines. Taken together, our results suggest that this Bax expression system might represent a useful gene therapy strategy when applied to the treatment of prostate cancer and its efficacy would be independent of the Bcl-2 status and androgen sensitivity of these cancers.

Adenoviridae↗

PubMedDate not supplied

Telomerase promoter-driven cancer gene therapy.

Human telomerase is highly active in more than 85% of primary cancers, regardless of their tissue origins, but not in most differentiated somatic tissues. Because of this, telomerase recently has become a popular target of anticancer therapy and has been used as a marker of cancer. Similarly, telomerase promoters, especially telomerase reverse transcriptase (TERT) promoter, have been zealously tested for targeted cancer gene therapy. In vitro and in vivo studies have demonstrated that the human TERT (hTERT) promoter is highly active in human and murine cancer cells but not in normal differentiated human cells, normal human CD34(+) progenitor cells, normal mouse fibroblasts, or normal mouse livers. Moreover, transcription factors can dramatically augment transgene expression from the hTERT promoter without the promoter losing its specificity. Thus far, telomerase promoters have been tested for targeted cancer gene therapy with proapoptotic, cytotoxic, and prodrug-activating genes and with replication-competent viral vectors. Here, we review recent achievements in telomerase promoter-based targeted cancer gene therapy.

Adenoviridae↗

PubMedDate not supplied

Tumor-suppressing gene therapy.

Tumor-suppressor genes play pivotal roles in maintaining genome integrity and in regulating cell proliferation, differentiation, and apoptosis. Their loss-of-function mutations are related directly to tumorigenesis. Thus, use of tumor-suppressor genes as anticancer therapeutics has been investigated rigorously in both experimental and clinical researches. Transfer of various tumor-suppressor genes directly to cancer cells has been demonstrated to suppress tumor growth via induction of apoptosis and cell-cycle arrest and, in some cases, with evidence for bystander effects. Various studies also have shown that combination of tumor-suppressor gene therapy with conventional anticancer therapy can yield synergistic therapeutic benefits. Clinical trials with tumor-suppressor genes, especially the p53 gene, have demonstrated that the treatment is well tolerated, and; favorable clinical responses, including a pathologically complete responses, have been observed in a subset of patients with advanced disease or with cancers resistant to conventional therapy. Yet, current gene replacement approaches in cancer gene therapy must be improved if they are to have a broader clinical impact. Efficient systemic gene delivery systems will be required ultimately for treatment of metastatic disease. In this review, we have recently summarized achievements in tumor-suppressor gene therapy with a focus on the p53 gene.

Animals↗

PubMedDate not supplied

Gene therapy in colon cancer cells with a fiber-modified adenovector expressing the TRAIL gene driven by the hTERT promoter.

BACKGROUND: Repeated administration of adenoviral vectors can lead to cell resistance, probably because of the initial coxsackie-adenovirus receptor (CAR). Modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence can overcome resistance. We constructed an adenoviral vector with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD), and tested its antitumor activity in 5 colon carcinoma cell lines. MATERIALS AND METHODS: Colon cancer cells were infected with Ad/CMV-GFP (vector control), Ad/gTRAIL (positive control) and Ad/TRAIL-FIRGD. PBS was used as a control. Cell viability was determined by proliferation assay. Cell-cycle analysis and quantification of Caspase-8 and TRAIL were used to identify apoptosis. RESULTS: Treatment with Ad/TRAIL-F/RGD resulted in significantly less cell viability, increased Caspase-8 and TRAIL activity, and a greater apoptotic fraction than treatment with PBS or Ad/CMV-GFP. CONCLUSION: The adenoviral vector Ad/TRAIL-F/RGD could become a potent therapeutic agent for the treatment of colon carcinoma.

Adenoviruses, Human↗