Botryoid-type of embryonal rhabdomyosarcoma of renal pelvis in a young woman.

A 22-year-old woman presented with three weeks history of intermittent left loin pain, on radiological evaluation by U\S and MRI revealed left renal pelvic mass, ureterorenoscopy and biopsy taken, but couldn't reveal definitive diagnosis other than presence of a malignant process. Left nephroureterctomy was performed. Grossly there is a polypoid mass attached to the upper pole of the kidney by stalk. Light microscopic examination and immunohistochemical staining confirm a diagnosis of Botryoid-type of embryonal rhabdomyosarcoma. Treatment and follow up to 1 year is mentioned. Reviewing the literature the presented case is the second of this tumor in adult renal pelvis.

Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv.

Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of scFv in situ. To circumvent these limitations, we have developed an approach to express scFv in vivo. An anti-erbB2 scFv was engineered for secretion by eukaryotic cells. The secreted scFv could bind to its target and specifically suppress cell growth of erbB2-positive cells in vitro. Adenoviral vectors expressing the cDNA for the secretory scFv likewise could induce target cells to produce an anti-tumor anti-erbB2 scFv. In vivo gene transfer via the anti-erbB2 scFv encoding adenovirus also showed anti-tumor effects. Thus, by virtue of engineering a secreted version of the anti-tumor anti-erbB-2 scFv, and in vivo expression via adenoviral vector, effective concentrations of scFv were achieved. In vivo gene transfer clearly represents a powerful means to realize effective scFv-based approaches. This method will likely have applicability for a range of disorders amenable to targeted therapeutic approaches.

Bax-induced apoptosis as a novel gene therapy approach for carcinoma of the cervix.

OBJECTIVE: The transfer of tumor suppressor genes has been shown to revert the malignant phenotype. In this regard, bax is a pro-apoptotic molecule that also functions as a tumor suppressor. The purpose of this study was to evaluate bax as a gene therapeutic in the context of cervical cancer. METHODS: Efficiency of viral transduction in cervical cancer cell lines and primary cervical cancer cells was evaluated with an adenoviral vector encoding green fluorescent protein and luciferase, respectively. We generated a recombinant adenoviral vector that encodes the bax gene under inducible conditions. To this end, expression of this pro-apoptotic gene was controlled by a Cre-LoxP system. Following infection with the recombinant bax adenovirus, the viability of cervical cancer cell lines and primary cervical cancer cells was evaluated using crystal violet staining and FACS analysis. Apoptotic cell death was monitored using annexin V staining. RESULTS: High levels of viral infection were observed in all cervical cancer cell lines (>85%) and primary cervical cancer cells. Significant cytotoxicity was seen in all cervical cancer cells lines and, more importantly, patient-derived primary cervical cancer cells. Moreover, bax-mediated cell death occurred via an apoptotic pathway. CONCLUSIONS: Our results indicate that a bax recombinant adenoviral vector causes cell death mediated via an apoptotic pathway in multiple cervical cancer cell lines and primary cervical cancer cells. These data suggest that bax may be a candidate for human gene therapy in the setting of cervical carcinoma.

Strategies to accomplish targeted expression of transgenes in ovarian cancer for molecular therapeutic applications.

PURPOSE: The purpose of the study was to determine the capability of the midkine (MK) and cycooxygenase-2 (cox-2) gene promoter regions to function as tumor-specific promoters for use in targeted gene therapy of ovarian cancer. EXPERIMENTAL DESIGN: Established and primary ovarian cancer and mesothelial cells were transduced by adenoviral vectors containing a reporter or thymidine kinase gene expressed under the control of the MK, cox-2, or cytomegalovirus (CMV) promoters. SCID or C57BL/6 mice were injected i.p. with these same vectors. In vitro reporter gene expression and cellular cytotoxicity was determined using luciferase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Acute toxicity in vivo was assessed by histological evaluation of harvested tissues. RESULTS: Consistent activation of the MK and cox-2 promoters was noted in all of the ovarian cancer cell lines in addition to primary ovarian cancer cells. In contrast, reduced reporter activity was reported in mesothelial cells transduced with adenoviruses containing the test promoters, which was especially apparent for the cox-2 promoter. Additionally, the cox-2 promoter exhibited significantly lower reporter gene levels in liver and peritoneum than the control promoter in in vivo experiments. Tumor-cell killing induced by Adcox-2 MTK was comparable to that observed with AdCMVTK. However, a clear differential toxicity pattern was observed in favor of animals treated with Adcox-2 MTK when compared with controls. CONCLUSIONS: These data clearly demonstrate that the transcriptional control afforded by the cox-2 promoter is tumor-specific and is able to mitigate associated toxicity in normal tissue while maintaining therapeutic efficacy in the context of an ovarian cancer molecular chemotherapeutic approach.

An immunomagnetic-based method for the purification of ovarian cancer cells from patient-derived ascites.

OBJECTIVE: Primary ovarian cancer cells obtained from fresh tumor have many advantages over established cell lines. Therefore, a procedure for the specific and efficient purification of such neoplastic cells is critical. We report an effective immunomagnetic method for the isolation of tumor cells from the ascitic fluid of patients diagnosed with ovarian adenocarcinoma. METHODS: This procedure incorporates the use of monoclonal antibody (mAb) CC49, which recognizes the tumor-associated glycoprotein 72 (TAG-72). TAG-72 is highly expressed on ovarian tumor cell surfaces with little or no reactivity with normal tissues. Also used in this protocol are immunomagnetic beads, which bind to the CC49 mAb via a secondary antibody. When ovarian cancer cells adhere to the magnetic beads, a magnetic field is used to separate the tumor cells from all other cellular components. RESULTS: Using ascitic fluid from five patients, we found that preparations before purification contained between 38 and 52% neoplastic cells. Using our method, we produced preparations that were between 63 and 96% pure for cancer cells, thus obtaining an average increase in tumor cell enrichment of 86%. CONCLUSION: We, therefore, believe this method is preferable for producing high yields of pure ovarian neoplastic cells. We are now employing this technique in our laboratory to provide a stringent and pure template for our studies on gene transfer to primary ovarian cancer cells.

Genetically modified CD34+ cells exert a cytotoxic bystander effect on human endothelial and cancer cells.

We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34+ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34+ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34+ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34+ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34+ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34+ cells and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34+ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34+ cells and total cell killing. For example, when 50% of CD34+ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of all cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80% of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34+ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.

Adenoviral-mediated suicide gene therapy for ovarian cancer.

The purpose of this phase I study was to determine the potential efficacy of adenoviral-mediated suicide gene therapy in women with recurrent ovarian cancer. Fourteen patients were treated intraperitoneally with herpes simplex virus-thymidine kinase (HSV-TK)-encoding adenovirus (AdHSV-TK) in dosages ranging between 1x10(9) and 1x10(11) pfu. Beginning 2 days later, ganciclovir (GCV) was administered intravenously at a dose of 5 mg/kg bid for 14 days. Transient vector-associated fever was experienced by 4 of 14 (29%) treated patients. Other possible vector-associated constitutional symptoms, abdominal pain, and gastrointestinal symptoms were experienced by 6 of 14 (43%) treated patients. No other dose-limiting vector-specific side effects were noted. Of the 13 patients evaluable for response, 5 (38%) had stable disease and 8 (62%) had evidence of progressive disease. Molecular analysis of evaluable ascites samples demonstrated the presence of transgene DNA and RNA in most patients 2 days following Ad HSV-TK administration. Ten of 11 evaluable patients had an increase in anti-adenovirus antibody titer. These results suggest that treatment with AdHSV-TK in combination with GCV is feasible in the context of human ovarian cancer and tolerated at the dosages studied.

Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target.

Intracellular single-chain antibodies (scFvs) have emerged as a powerful method to knock out expression of oncoproteins. We have demonstrated previously that scFvs directed against a variety of molecular targets induce specific toxicity in tumor cells. Recently, the utility of an anti-erbB-2 scFv has predicated its evaluation in a phase I gene therapy clinical trial. The utility of scFv as an intrabody is closely linked to its interaction with a target, limiting the contribution of the latter to the neoplastic phenotype. In this study, we sought to determine whether improvement in the affinity of the scFv for its cognate target could improve the efficiency of intrabody-mediated oncoprotein knockout. We compared in erbB-2-positive and -negative tumor cells the function of plasmids encoding a newly developed C6.5 anti-erbB-2 scFv, which has a 1000-fold higher affinity, with our original e23 anti-erbB-2 scFv. Intracellular scFv expression, target binding, and tumor cell cytotoxicity were found to be similar in all conditions tested, including dose-response studies with limiting dilutions of the scFv. On this basis, we have concluded that the antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target.

An adenovirus encoding proapoptotic Bax induces apoptosis and enhances the radiation effect in human ovarian cancer.

Overexpression of proapoptotic Bax favors death in cells resistant to ionizing radiation. We hypothesized that expression of Bax via adenoviral-mediated gene delivery could sensitize radiation-refractory cells to radiotherapy. An inducible Bax recombinant adenovirus (Ad/Bax) had been generated using the Cre/loxp system. Human ovarian cancer cell lines and primary, patient-derived cancer cells from ascites were irradiated and infected with the Ad/Bax and an expression-inducing vector, Ad/Cre. Cell death was evaluated by crystal violet staining, fluorescence-activated cell sorter analysis of Annexin V, and colony formation assay (cell lines only). To further characterize the mechanism of death, cell morphology was examined by nuclear staining with Hoechst 33258. Lastly, to evaluate the capacity of the combined treatment to inhibit tumor growth, mice were injected subcutaneously with ovarian cancer cells exposed to Bax, radiation therapy (RT), or both, and tumor size was measured periodically. Infection of the cancer cell lines and primary cells with both Ad/Bax and Ad/Cre significantly enhanced sensitivity to ionizing radiation, achieving high levels of cell killing in short-term assays. In addition, the combination of Bax and radiotherapy reduced the survival fraction of cell lines 2 logs in standard colony-forming assays. Investigation into the involved mechanism suggests that Bax-mediated radiosensitization occurs through both apoptosis and necrosis pathways. Further, mice subcutaneously injected with ovarian tumor cells previously treated with radiation, or with radiation and irrelevant viruses, consistently developed tumor nodules. In addition, approximately 80% of injections were followed by tumor formation after treatment with Ad/Bax and Ad/Cre alone. In contrast, tumor formation was completely inhibited after combined treatment with Ad/Bax and Ad/Cre and radiation. Augmentation of the effect of radiotherapy on human ovarian cancer cells and primary cancer cells from patients via a recombinant adenovirus encoding Bax is feasible.

A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial.

The purpose of this Phase I study was to determine the feasibility of using an anti-erbB-2-encoding adenovirus (Ad21) to treat erbB-2-overexpressing ovarian cancer. Recurrent ovarian cancer patients were treated i.p. with Ad21 in dosages ranging from 1 x 10(9) to 1 x 10(11) pfu. Patients were monitored after treatment for evidence of clinical toxicity and efficacy. Peritoneal aspirates and serum samples were obtained to assess for evidence of gene transfer/expression, for generation of wild-type vector, and antiadenoviral humoral response. Fifteen patients were treated per study specifications. Treatment-specific grade 1/2 fever was experienced by 9 of 15 (60%) patients. Other transient grade 1/2 constitutional, pain, and gastrointestinal symptoms were also experienced. No dose-limiting vector-related toxicity was experienced. Of 13 patients evaluable for response, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease. One patient with nonmeasurable disease normalized her CA125 at the 8-week evaluation, and one patient with nonmeasurable disease remained without clinical evidence of disease for 6 months after treatment. PCR analysis of peritoneal aspirates demonstrated the presence of Ad21 in 84.6%, 84.6%, and 61.6% of evaluable specimens at days 2, 14, and 56 after treatment, respectively. No wild-type virus was detected. Reverse transcription-PCR analysis demonstrated expression of the anti-erbB-2 sFv-encoding gene in 10 of 14 evaluable patients at day 2. Five of six evaluable patients had an increase in antiadenovirus antibody titer. This study suggests that adenoviral-mediated gene therapy using an anti-erbB-2-directed intrabody is feasible in the context of human ovarian cancer.

Pro-apoptotic treatment with an adenovirus encoding Bax enhances the effect of chemotherapy in ovarian cancer.

BACKGROUND: Tumor cell heterogeneity and resistance to chemotherapy-mediated cell death are major obstacles in cancer therapy. It has been reported that expression of the pro-apoptotic molecule Bax can induce cell death or sensitize tumor cells to chemotherapy in stable cell clones derived from tumor cells. However, these studies are limited in that they cannot represent the heterogeneity of cancer cells observed in vivo. In this study, we have further explored the therapeutic potential of Bax. METHODS: Using an inducible recombinant Bax adenovirus, we screened a panel of ovarian cancer cell lines and primary patient-derived ovarian tumor cells for their sensitivity to Bax-mediated cytotoxicity. Apoptotic cell death was evaluated qualitatively with Hoechst staining and quantitatively with MTS and Annexin V-based assays. Endogenous levels of both Bcl-2 and Bax protein and p53 status were evaluated. The potential of bax to sensitize ovarian cancer lines to chemotherapy was also tested. Dose-response curves were generated to evaluate cell death. RESULTS: Overexpression of Bax directly induced apoptosis in both ovarian cancer cell lines and the patient-derived primary cancer cells. However, the sensitivity of these cells to Bax varied and appeared to be independent of both the status of p53 and the endogenous levels of bcl-2 or Bax, critical molecules in the apoptotic pathway. Importantly, overexpression of Bax significantly enhanced chemotherapy-induced cytotoxicity in both established cell lines and primary ovarian carcinoma cells. CONCLUSIONS: These studies suggest that overexpression of Bax alone or in combination with chemotherapy may provide a means to overcome the problems imposed by the heterogeneous nature of tumors, ultimately augmenting the efficacy of chemotherapy in patients suffering from ovarian cancer.

Genetically modified CD34+ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model.

To develop a cellular vehicle able to reach systemically disseminated areas of angiogenesis, we sought to exploit the natural tropism of circulating endothelial progenitor cells (EPCs). Primate CD34+ EPCs were genetically modified with high efficiency and minimal toxicity using a non-replicative herpes virus vector. These EPCs localized in a skin autograft model of angiogenesis in rhesus monkeys, and sustained the expression of a reporter gene for several weeks while circulating in the blood. In animals infused with autologous CD34+ EPCs transduced with a thymidine kinase-encoding herpes virus, skin autografts and subcutaneous Matrigel pellets impregnated with vascular growth factors underwent necrosis or accelerated regression after administration of ganciclovir. Importantly, the whole intervention was perfectly well tolerated. The accessibility, easy manipulation, lack of immunogenicity of the autologous CD34+ cell vehicles, and tropism for areas of angiogenesis render autologous CD34+ circulating endothelial progenitors as ideal candidates for exploration of their use as cellular vehicles when systemic gene delivery to those areas is required. Gene Therapy (2000) 7, 43-52.

Gene therapy for carcinoma of the breast: Pro-apoptotic gene therapy.

The dysregulation of apoptosis contributes in a variety of ways to the malignant phenotype. It is increasingly recognized that the alteration of pro-apoptotic and anti-apoptotic molecules determines not only escape from mechanisms that control cell cycle and DNA damage, but also endows the cancer cells with the capacity to survive in the presence of a metabolically adverse milieu, to resist the attack of the immune system, to locally invade and survive despite a lack of tissue anchorage, and to evade the otherwise lethal insults induced by drugs and radiotherapy. A multitude of apoptosis mediators has been identified in the past decade, and the roles of several of them in breast cancer have been delineated by studying the clinical correlates of pathologically documented abnormalities. Using this information, attempts are being made to correct the fundamental anomalies at the genetic level. Fundamental to this end are the design of more efficient and selective gene transfer systems, and the employment of complex interventions that are tailored to breast cancer and that are aimed concomitantly towards different components of the redundant regulatory pathways. The combination of such genetic modifications is most likely to be effective when combined with conventional treatments, thus robustly activating several pro-apoptotic pathways.

Patulin-induced inhibition of protein synthesis in hepatoma tissue culture.

Patulin is a mycotoxin produced by several species of fungi and is commonly found in fruits. It is regulated in several countries at a tolerance level of 50 micrograms/Kg. We investigated its ability to inhibit cell growth in hepatoma tissue culture and its ability to inhibit protein synthesis. It was found to be cytostatic at concentration of 1 microgram/mL (6.4 microM). It inhibits protein synthesis by two mechanisms: inhibition of amino acid uptake into the cell and their incorporation into proteins. The former mechanism appears to be more significant than the latter. This is consistent with previous work showing the ability of patulin to perturb plasma membrane function.

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin.

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.