Biomedical subjects

Meng Yang

Publications and source records attributed to Meng Yang.

49 records · Page 3Linked to original sources

PubMed2002

High efficiency genetic modification of hair follicles and growing hair shafts.

A technique for genetic modification of hair follicles was developed which results in efficient alteration of the hair shaft phenotype. High-level in vivo transgene expression was maintained in hair follicles such that growing hair shafts were phenotypically altered. Mouse anagen skin fragments, maintained in histoculture, were genetically modified at high efficiency with adenoviral-GFP. The histocultured skin fragments were treated with collagenase which made hair follicles accessible to the adenoviral GFP gene, allowing high-efficiency transduction. These skin fragments were subsequently grafted on to nude mice where GFP was readily visualized in as many as 75% of hair follicles. Most follicles produced GFP-fluorescent growing hair shafts. This technique has produced efficient genetic modification of the hair shaft.

Adenoviridae↗

PubMed2002

A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy.

p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16(INK4a). Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone-respond poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16(INK4a), and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.

Animals↗

PubMed2002

Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model.

Mouse tumor models have undergone profound improvements in the fidelity of emulating human disease. Replacing ectopic s.c. implantation with organ-specific orthotopic implantation reproduces human tumor growth and metastasis. Strong fluorescent labeling with green fluorescent protein along with inexpensive video detectors, positioned externally to the mouse, allows the monitoring of details of tumor growth, angiogenesis, and metastatic spread. However, the sensitivity of external imaging is limited by light scattering in intervening tissue, most especially in skin. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold. The observable depth of tissue is thereby greatly increased and many tumors that were previously hidden are now clearly observable. This report presents tumor images and related quantitative growth data previously impossible to obtain. Single tumor cells, expressing green fluorescent protein, were seeded on the brain image through a scalp skin-flap. Lung tumor microfoci representing a few cells are viewed through a skin-flap over the chest wall, while contralateral micrometastases were imaged through the corresponding skin-flap. Pancreatic tumors and their angiogenic microvessels were imaged by means of a peritoneal wall skin-flap. A skin-flap over the liver allowed imaging of physiologically relevant micrometastases originating in an orthotopically implanted tumor. Single tumor cells on the liver arising from intraportal injection also were detectable. Possible future technical developments are suggested by the image, through a lower-abdominal skin-flap, of an invasive prostate tumor expressing both red and green fluorescent proteins in separate colonies.

Animals↗

PubMed2002

Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.

We report here whole-body optical imaging, in real time, of genetically fluorescent pancreatic tumors growing and metastasizing to multiple sites in live mice. The whole-body optical imaging system is external and noninvasive. Human pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express high-levels of the Aequorea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell lines were surgically orthotopically implanted as tissue fragments in the body of the pancreas of nude mice. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. Intravital images in the opened animal confirmed the identity of whole-body images. The whole-body images were used for real-time, quantitative measurement of tumor growth in each of these organs. Intravital imaging was used for quantification of growth of micrometastasis on the liver and stomach. Whole-body imaging was carried out with either a trans-illuminated epi-fluorescence microscope or a fluorescence light box, both with a thermoelectrically cooled color CCD camera. The simple, noninvasive, and highly selective imaging made possible by the strong GFP fluorescence allowed detailed simultaneous quantitative imaging of tumor growth and multiple metastasis formation of pancreatic cancer. The GFP imaging affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals without the need for anesthesia, substrate injection, contrast agents, or restraint of animals required by other imaging methods. The GFP imaging technology presented in this report will facilitate studies of modulators of pancreatic cancer growth, including inhibition by potential chemotherapeutic agents.

Animals↗

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

Dissecting p53 tumor suppressor functions in vivo.

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.

Aneuploidy↗

PubMed2002

Optically imageable metastatic model of human breast cancer.

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease.

Adult↗

PubMed2002

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression.

There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein (GFP) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFP-expressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.

Cell Division↗

PubMed2002

Parathyroid hormone-related protein as a novel tumor marker in pancreatic adenocarcinoma.

INTRODUCTION: Parathyroid hormone-related protein (PTHrP) can act as an oncoprotein to regulate the growth and proliferation of many common malignancies, including pancreatic cancer. Previous studies have shown that PTHrP is produced by human pancreatic cancer cell lines, can be shown in the cytoplasm and nucleus of paraffin-embedded pancreatic adenocarcinoma tumor specimens, and is secreted into the media of cultured pancreatic adenocarcinoma cells. We hypothesized that PTHrP could serve as a tumor-marker for growth of pancreatic cancer in vivo. AIM AND METHODOLOGY: To test this hypothesis, we used an orthotopic model developed in our laboratory of the PTHrP-producing human pancreatic cancer line, BxPC-3. This tumor was stably transduced with green fluorescence protein (GFP) to facilitate visualization of tumor growth and metastases. At early (5 weeks) and late (13 weeks) time points after surgical orthotopic implantation, serum PTHrP was measured and primary and metastatic tumor burden was determined for each mouse by assessing GFP expression. RESULTS: By 5 weeks after surgical orthotopic implantation (early group), the mean serum PTHrP level was 33.3 pg/mL. In contrast, by 13 weeks after surgical orthotopic implantation (late group), the mean serum PTHrP level increased to 158.5 pg/mL. These differences were highly significant (p < 0.001, Student t test). Numerous metastatic lesions were readily visualized by GFP in the late group. Serum PTHrP levels measured by immunoassay correlated with primary pancreatic tumor weights and serum calcium levels (p <0.01). PTHrP levels were not detectable (<21 pg/mL) in any of the 10 control mice with no tumor. Western blotting of BxPC-3-GFP tumor lysates confirmed the presence of PTHrP. BxPC-3-GFP tumor tissue stained with antibody to PTHrP. CONCLUSION: These results indicate that PTHrP can serve as a tumor marker in animal models of pancreatic cancer and may be a useful tumor marker for clinical pancreatic adenocarcinoma.

Adenocarcinoma↗

PubMedDate not supplied

Antimetastatic efficacy of oral 5-FU imaged by green fluorescent protein in real time.

The effectiveness of oral 5-FU in suppressing liver metastasis was assessed in a highly-metastatic mouse model. Doses of 20 and 25 mg/kg oral 5-FU significantly suppressed primary and metastatic tumor growth (p = 0.012). These inhibitory effects were more dramatic in suppressing liver metastasis (p = 0.0). The efficacy of 5-FU was visualized by whole-body fluorescence imaging of the green fluorescent protein-expressing tumor and its subsequent metastases. Toxicity was observed only in the 30 mg/kg dose. Furthermore, we showed that the non-toxic doses of 5-FU significantly prolonged survival in these animals. These data suggest the important clinical potential of oral 5-FU.

Administration, Oral↗

PubMedDate not supplied

Oral 5-FU is a more effective antimetastatic agent than UFT.

5-Fluorouracil (5-FU), a pyrimidine analog, is widely used to treat gastrointestinal and other cancers. In the present study, we compared the efficacy of oral 5-FU and the 5-FU prodrug, uracil plus tegafur (UFT), on liver metastasis in a highly metastatic mouse model. Genetic labeling of the tumor with green fluorescent protein (GFP) along with inexpensive video detectors, positioned external to the mouse, allowed the real-time monitoring of details of tumor growth, metastatic spread and drug response in this mouse model. 5-FU at 10 and 20 mg/kg significantly prolonged the survival time of treated animals compared with untreated controls (p=0.003 for 5-FU, 10 mg/kg; p=0.016 for 5-FU, 20 mg/kg). In contrast, UFT only showed a trend to increase survival (p=0.078). 5-FU at 10 mg/kg substantially prolonged the survival time compared to UFT (p=0.012). 5-FU (10 mg/kg) was also more effective in prolonging survival than Furtulon (5'-deoxy-5-fluorouridine, another 5-FU prodrug) (p=0.042). All control and UFT-treated animals died by day 45. In contrast, at 45 days, 5 out of 8 animals were alive in the 5-FU 10 mg/kg group, which was found to be the best treatment regimen in this study. 5-FU (10 mg/kg)-treated animals had a median survival time of 53 days compared to 26.5 in controls and 33.5 days for UFT. These results suggest the potential clinical superiority of oral 5-FU compared to UFT as an anti-metastatic agent. The data also suggest the lack of clinical need for complex and expensive prodrugs of 5-FU such as UFT.

Animals↗

PubMedDate not supplied

Visualizing portal vein metastatic trafficking to the liver with green fluorescent protein-expressing tumor cells.

Cell migration or trafficking is an integral aspect of cancer metastasis and is a target for development of novel antimetastatic therapy. Tumor cell trafficking has been a poorly understood phenomenon due to the inability to visualize the process. In this study, we visualized the trafficking of metastatic cells targeting the liver via the portal vein using green fluorescent protein (GFP)-expressing cancer cells. Within 72 h after transplantation of tumor cells, on the ascending colon in nude mice, metastasis was visualized ex vivo on a single-cell basis around the portal vein by GFP imaging. At this early time-point, a few cells were visualized trafficking to the liver via the portal vein. By post-implantation day-5, the caudate lobe of the liver was involved with trafficking metastatic cells. Metastasis around the portal vein increased more rapidly than those in other areas of the liver. By day-7 post-implantation, the right lateral lobe of the liver was involved with trafficking metastatic cells. By days-9 and -11, metastasis increased rapidly around the portal vein and then spread to other areas of the liver. These experiments demonstrate the critical role of the portal vein in metastasis to the liver.

Animals↗

PubMedDate not supplied

Color coding cancer cells with fluorescent proteins to visualize in vivo cellular interaction in metastatic colonies.

We report here in vivo visualization of cancer cell interaction using stable sublines of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma cells. These color-coded fibrosarcoma cell lines show similar cell proliferation and lung metastasis potential. The color-coded cells were mixed at a ratio of 1:1 and were injected into the tail vein of severe combined immunodeficient (SCID) mice. The resulting experimental lung metastases were simultaneously imaged and their ratio was determined by color-pixel analysis. Fluorescence color coding of cancer cells enables visualization of the interaction of cancer cells and can be used to distinguish cancer cells of any genotype or phenotype.

Animals↗