Biomedical subjects

S Chada

Publications and source records attributed to S Chada.

At least 19 recordsLinked to original sources

PubMed2006

Enhancement of adenoviral MDA-7-mediated cell killing in human lung cancer cells by geldanamycin and its 17-allyl- amino-17-demethoxy analogue.

Our previous studies demonstrated that adenovirus-mediated overexpression of melanoma differentiation-associated gene-7 (Ad-mda7) leads to rapid induction of double-stranded RNA-dependent protein kinase (PKR) and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. Here, we report that Ad-mda7 and the benzoquinone ansamycin geldanamycin (GA) interact in a highly synergistic manner to induce cell death in human lung cancer cells. Co-administration of Ad-mda7 and GA did not modify expression of MDA-7, and was not associated with further PKR induction and activation; instead the enhanced cytotoxicity of this combination was associated with inactivation of AKT by GA. By surface staining using anti-E-cadherin monoclonal antibody and flow cytometry, we found that treatment with the combination of Ad-mda7 and GA increased E-cadherin levels in these cancer cells. Ad-mda7 and GA cotreatment also inhibited lung cancer cell motility by increasing the beta-catenin/E-cadherin association. Moreover, combination of GA derivative 17-allyl-amino, 17-demethoxygeldanamycin (17AAG), with Ad-mda7 resulted in enhancement of cell death in A549 and H460 human lung cancer cells.

Adenoviridae↗

PubMed2006

Combinatorial synergy induced by adenoviral-mediated mda-7 and Herceptin in Her-2+ breast cancer cells.

The melanoma differentiation-associated gene-7 (mda-7) is a member of the interleukin-10 cytokine family and a novel tumor suppressor gene. Adenoviral-mediated mda-7 (Ad-mda7) gene transfer has tumor-specific growth inhibitory and proapoptotic effects in a broad spectrum of cancer cells. In breast cancer cells, adenoviral-induced mda-7 expression triggers antiproliferative effects by downregulation of survival signals, such as Bcl-2 and Akt. The anti-human epidermal growth factor receptor-2 (Her-2) monoclonal antibody, Trastuzumab (Herceptin), increases the sensitivity of Her-2/neu-overexpressing breast cancer cells to chemotherapeutic agents and radiotherapy. In this study, we evaluate the effects of treatment with Ad-mda7 and Herceptin combination therapy in a panel of Her-2/neu-overexpressing cell lines, and in established tumors in nude mice. Compared to individual treatments, the combination of Ad-mda7 and Herceptin elicits supra-additive antitumor activity in Her-2/neu-overexpressing tumor cell lines: increased cell death, cell cycle block and apoptosis. The Ad-mda7 and Herceptin interaction was shown to be synergistic by isobologram analysis. Ad-mda7 does not alter cell surface Her-2/neu levels, but the combination of Ad-mda7+Herceptin results in increased expression of cell surface E-cadherin with concomitant translocation of beta-catenin from the nucleus to the cell membrane. In vivo, the combination of Ad-mda7 and Herceptin showed significantly increased antitumor activity (P<0.003) against Her-2/neu-overexpressing tumors. These data suggest that the combination of Ad-mda7 with Herceptin may be a novel therapy for breast cancer patients whose tumors overexpress Her-2/neu. The observed synergistic effect may improve treatment options for otherwise poorly responsive, Her-2-positive, breast cancer patients.

Adenoviridae↗

PubMed2006

Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model.

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Adenoviridae↗

PubMed2006

mda-7 gene transfer sensitizes breast carcinoma cells to chemotherapy, biologic therapies and radiotherapy: correlation with expression of bcl-2 family members.

Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We have previously shown that adenoviral-mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report selective Ad-mda7-mediated p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (P<0.004) in multiple xenograft models. We then evaluated the combination of Ad-mda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), nonsteroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anticancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX upregulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (P<0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells.

Adenoviridae↗

PubMed2003

Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers.

Adenoviridae↗

PubMed2002

An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53.

P53-based immunization is an attractive approach to cancer immunotherapy due to the accumulation of p53 protein in tumor, but not in normal cells. However, it was not known whether immune response against self-protein (p53) could be generated in vivo. Mouse dendritic cells (DCs) were transduced with adenoviral construct containing murine full-length wild-type p53 (Ad-p53). Repeated immunizations with these cells protected 60% of mice against challenge with MethA sarcoma cells bearing point mutations in p53 gene. Activation of DCs via ligation of CD40 significantly improved the results of immunization: all mice were protected against MethA sarcoma. The treatment of MethA tumor-bearing mice with activated Ad-p53-transduced DCs showed complete tumor rejection in four out of six mice. The specificity of antitumor immune response was confirmed by CTL assay. The analysis of phenotype and function of DCs demonstrated that the effect of CD40 ligation on these cells was enhanced by their infection with Ad-p53. The level of neutralizing anti-adenovirus antibody was moderately elevated in these mice. No signs of autoimmune reaction were evident during detailed pathological evaluation of treated mice. These data demonstrate that activated Ad-p53-infected DCs are able to break tolerance to this protein and can be used in immunotherapy of cancer.

Adenoviridae↗

PubMed2001

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

Antigens, Neoplasm↗

PubMed2001

Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.

The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.

Apoptosis↗

PubMed2001

Gene therapy. Therapeutic approaches and implications.

The present article is an overview of gene therapy with an emphasis on different approaches and its implications in the clinic. Genetic interventions have been applied to the diagnosis of and therapy for an array of human diseases. The initial concept of gene therapy was focused on the treatment of genetic diseases. Subsequently, the field of gene therapy has been expanded, with a major focus on cancer. Although the results of early gene therapy-based clinical trials have been encouraging, there is a need for gene delivery vectors that feature reduced immunogenicity and improved targeting ability. The results of phases I/II clinical trials have suggested the important role of gene therapy as a versatile and powerful treatment tool, especially for human cancers. One reasonable expectation is that performing gene therapy at an earlier stage in the disease process or for minimal residual disease may be more advantageous.

Journal Article↗

PubMed2001

Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients.

Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.

Adenoviridae↗

PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗

PubMed2000

Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro.

The melanoma differentiation-associated gene-7 (mda-7), cloned from a human melanoma cell line H0-1, is known to induce tumor cell-selective growth inhibition in breast cancer cells in vitro and loss of tumorigenicity ex vivo. Yet, the mechanisms underlying these effects are still unknown. Therefore, we investigated these mechanisms on the molecular level in human non-small cell lung carcinoma (NSCLC) cells in vitro. Overexpression of mda-7 protein by Ad-mda-7 significantly suppressed proliferation and induced G2/M cell cycle arrest in wild-type p53 (A549, H460), and p53-null (H1299) non-small cell lung cancer cell lines, but not in normal human lung fibroblast (NHLF) cells. p53, Bax, and Bak protein expression was up-regulated in wild-type p53 tumor cell lines, but not in p53-null cells, suggesting that an intact p53 pathway was required for Bax and Bak induction. However, in all three cancer cell lines tested, activation of the caspase cascade and cleavage of poly(ADP-ribose) polymerase (PARP) appeared to be independent of the p53 mutational status. Together, these results suggest that apoptosis may be induced via multiple pathways by Ad-mda-7 in lung cancer cells and that Ad-mda-7 has the potential to become a novel therapeutic for clinical cancer gene therapy. Gene Therapy (2000) 7, 2051-2057.

Adenoviridae↗

PubMed1999

Radiosensitization of human tumor cell lines induced by the adenovirus-mediated expression of an anti-Ras single-chain antibody fragment.

The expression of activated ras genes has been implicated as a contributing factor to the radioresistance of tumor cells. As a strategy for compromising Ras protein activity and potentially enhancing the radiosensitivity of tumor cells, we have investigated the application of the AV1Y28 adenovirus, which expresses a single-chain antibody fragment directed against p21 Ras proteins. The ability of AV1Y28 transduction to modulate radioresponse was investigated using four human tumor cell lines--U251 glioblastoma, MIA PaCa-2 pancreatic carcinoma, and the colon carcinomas SW620 and HT29. Cultures were exposed to sufficient levels of AV1Y28 to transduce more than 90% of the cells; 24 h later, cultures were exposed to ionizing radiation, and clonogenic cell survival was determined. Tumor cell survival was reduced by 40-50% when the tumor cell lines were exposed to AV1Y28 only. In addition, for each tumor cell line, AV1Y28 exposure enhanced the level of radiation-induced cell killing. Dose enhancement factors at a surviving fraction of 0.1 ranged from 1.3 to 1.5. Furthermore, for each of the cell lines, the surviving fraction at 2 Gy was significantly reduced by AV1Y28 exposure. In contrast to the results seen in tumor cells, the radiosensitivity of a normal human fibroblast cell line was not affected by AV1Y28. These data indicate that this anti-Ras adenovirus enhances the radiosensitivity of tumor cells but does not affect the radiosensitivity of normal cells.

Adenoviridae↗

PubMed1999

Chronic aortic counterpulsation with latissimus dorsi in heart failure: clinical follow-up.

BACKGROUND: Dynamic aortomyoplasty is an alternative technique to heart transplantation. The goal of our study was to evaluate the benefits of aortic counterpulsation obtained by dynamic thoracic aortomyoplasty in patients with heart failure refractory to pharmacologic treatment and contraindications to heart transplant or cardiomyoplasty. METHODS: In this study we compared preoperative and postoperative data from five out of six carefully selected patients who were treated with dynamic thoracic aortomyoplasty. This surgical technique wraps the right latissimus dorsi muscle flap around the ascending aorta. This muscle flap was electrically stimulated during diastole, following a muscle-conditioning protocol, to obtain diastolic augmentation. At the 6-month follow-up period we evaluated, invasively and noninvasively, the hemodynamic and clinical effects of aortomyoplasty. RESULTS: We observed a significant decrease in the number of hospitalizations (P = 0.01), NYHA functional class (P = 0.01), cardiothoracic ratio (P = 0.02), right ventricular diameter (P = 0.03), left atrial diameter (P = 0.04), and pulmonary artery systolic pressure (P = 0.04); and a significant increase in the 6-minute walking test (P = 0.01), cardiac index (P = 0.04), noninvasive evaluation of diastolic augmentation (P = 0.01), left ventricular shortening fraction (P = 0.01), and radioisotopic left ventricular ejection fraction (P = 0.02). We also found a nonsignificant decrease in the left ventricular diameter (P = 0.08) and wedge pressure (P = 0.19); and a nonsignificant increase in peak oxygen consumption (P = 0.13). CONCLUSIONS: Dynamic thoracic aortomyoplasty in heart failure resulted in an important improvement of hemodynamic parameters, heart functional data, and clinical functional class, when comparing preoperative data with the 6-month follow-up data.

Adult↗

PubMed1999

Intra-articular IL-4 gene therapy in arthritis: anti-inflammatory effect and enhanced th2activity.

Gene therapy has been explored as a potential method for treating chronic inflammatory diseases such as rheumatoid arthritis. To determine the efficacy of intra-articular IL-4 gene therapy in an animal model of arthritis using a retroviral vector, a retrovirus encoding rat IL-4 (DA-IL-4) was engineered, purified and concentrated to high titer (>/=109 CFU/ml). Infectivity and expression levels were demonstrated in vitro using cultured fibroblast-like synoviocytes. Efficacy was evaluated in the rat adjuvant arthritis model. DA-IL-4 or DA-beta-gal retrovirus was injected into the intra-articular joint space of the right ankle on day 12 after immunization. Three days after joint injection, the injected paw contained increased levels of IL-4 compared with control or with the contralateral uninjected paw, demonstrating successful transgene expression. Surprisingly, 8 days after treatment IL-4 levels continued to increase in the injected and contralateral paw compared with DA-beta-gal-treated animals. Serum IL-4 levels were also elevated in DA-IL-4-treated rats. RT-PCR studies demonstrated that the transgene was expressed in the injected ankle but not in the contralateral joint. IL-4 gene therapy resulted in a significant reduction in paw swelling and decreased radiographic evidence of bone destruction. This is the first demonstration of successful intra-articular retroviral gene treatment using a therapeutic gene. In addition to its anti-inflammatory effect, this study supports the potential application of intra-articular gene therapy as a method for enhancing systemic Th2 function.

Animals↗

PubMed1999

Dendritic cells transduced with wild-type p53 gene elicit potent anti-tumour immune responses.

In this study we have tested the concept of using wild-type p53 gene for immunotherapy of cancer. Dendritic cells (DC) were transduced with a human wild-type p53 containing recombinant adenovirus (Ad-p53). About a half of DC transduced with this virus expressed p53 protein by FACS analysis 48 h after infection. Mice immunized twice with Ad-p53 DC developed substantial cytotoxic T lymphocyte (CTL) responses against tumour cells expressing wild-type and different mutant human and murine p53 genes. Very low CTL responses were observed against target cells infected with control adenovirus (Ad-c). Immunization with Ad-p53 provided complete tumour protection in 85% of mice challenged with tumour cells expressing human mutant p53 and in 72.7% of mice challenged with tumour cells with murine mutant p53. Treatment with Ad-p53-transduced DC significantly slowed the growth of established tumours. Thus, DC transduced with wild-type p53 may be a promising new tool for the immunotherapy of cancer.

Adenoviruses, Human↗

PubMed1998

Adenovirus-mediated wild-type p53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer [seecomments].

BACKGROUND: The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. METHODS: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. RESULTS: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. CONCLUSIONS: Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.

Adenocarcinoma↗

PubMed1998

Direct synovial gene transfer with retroviral vectors in rat adjuvant arthritis.

OBJECTIVE: To evaluate the feasibility of direct in vivo gene transfer in an animal model of arthritis using a retroviral vector. METHODS: The timing and dose of retroviral vector was examined using very high titer retroviral vector (> or = 10(9) CFU) in rat adjuvant arthritis. Retroviral vector expressing beta-galactosidase (beta-gal) or vehicle alone was injected into the right ankle of rats with adjuvant arthritis. Ankles were injected either on Day 7 (pre-arthritis), Day 10 (early arthritis), Day 15 (accelerating arthritis), or Day 28 (chronic arthritis) after adjuvant immunization. Joints were harvested 3 days later and extracts were assayed for beta-gal activity. RESULTS: Synovial beta-gal expression was minimal in the Day 7 group and elevated in the Day 10, Day 15, and Day 28 groups. Gene transfer with retroviral vector did not exacerbate the local inflammatory response. Minimal or no beta-gal expression was observed in the contralateral uninjected paw or in the spleen, lung, liver, and kidneys. Frozen sections of retroviral vector injected joints were stained with X-gal and revealed transduced cells in the lining and superficial sublining layers. To determine the longevity of gene expression, ankle joints were injected with vector on Day 15 post-adjuvant, harvested, and assayed for beta-gal activity for up to 49 days after injection. Expression of the enzyme peaked from Day 3 to 7 and was still readily detected up to 49 days after retrovirus infection. CONCLUSION: This is the first report of successful direct in vivo gene transfer in the rat adjuvant arthritis model using a retroviral vector. Appropriate timing of administration and very high titer retroviral vector preparations are key determinants of adequate gene transduction.

Animals↗