Laminin promotes rabbit neutrophil motility and attachment.

Polymorphonuclear neutrophils (PMN) traverse basement membrane to reach sites of infection. We have studied the role of laminin, a specific basement membrane component, in this process using three assay systems. In the Boyden chamber, laminin was found to stimulate chemotaxis of neutrophils while fibronectin did not. Co-incubation of cells with antibody to laminin blocked this chemotaxis, while antibody to fibronectin was without effect. In the human amnion system, neutrophils were shown to penetrate through the tissue when the peptide chemoattractant f-Met-Leu-Phe was placed on the opposing side. Antibody to laminin, but not to fibronectin, blocked this penetration. In an attachment assay system, laminin, but not fibronectin, was found to increase dispase-treated neutrophil attachment to type IV (basement membrane) collagen-coated plastic and to a plastic substrate itself. Electrophoretic analysis of PMN extract indicated the presence of laminin, and indirect immunofluorescence suggested that laminin is localized on the surface of the neutrophils. These data suggest that PMN can bind laminin on their cell surfaces, use laminin to attach to basement (type IV) membrane collagen, and migrate toward a gradient of laminin. These properties may be important for the passage of neutrophils from the circulation to sites of infection.

Targeted tumor killing via an intracellular antibody against erbB-2.

Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis.

A three-dimensional assay for measurement of viral-induced oncolysis.

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.

Breast cancer metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice.

This study sought to determine if weekly X-ray exposure affected breast cancer cell metastasis to bone and to also evaluate the use of bioluminescent imaging (BLI) and microSPECT for detection of metastatic bone lesions. Five week old nude mice were randomly assigned to the CT exposed (n = 7) and no CT exposure (n = 6) treatment groups. Mice received an intracardiac injection of MDA-MB-435 human breast cancer cells transduced with luciferase, or a sham injection (saline). The CT exposed group of mice received CT irradiation once a week for 5 weeks. All mice underwent weekly BLI and select mice received Tc-99m-MDP followed by microSPECT imaging after 5 weeks. Pathological evaluation and histomorphometry were used to assess the affect of CT X-rays on bone metastasis and to evaluate BLI. BLI results found no significant difference in metastasis between animals that received CT and those that did not (P > 0.05); however, histomorphometry of the knee joints revealed a significant increase (P = 0.029) in tumor area of the leg bones in mice that received CT exposure (60% +/- 7%) compared to animals that did not receive CT scans (33% +/- 8%). Compared to histological analysis, BLI of the leg and spine was determined to have excellent sensitivity (100%), good specificity (80-90%) and accuracy (90-96%), a positive predictive value of 81-93% and a 100% negative predictive value. Thus, multi-modality imaging techniques can be very useful for monitoring bone metastasis, however microCT X-rays should be used judiciously in order to limit irradiation that may stimulate increased metastasis to specific regions of the skeleton. MicroSPECT imaging did not detect metastatic lesions in the legs of these young nude mice.

Evolving gene therapy approaches for osteosarcoma using viral vectors: review.

This review begins with an introduction to the malignant bone tumor, osteosarcoma [OS] and then moves to a discussion of the commonly used vectors for gene transfer. We first briefly highlight non-viral vectors including polymeric and liposomal delivery systems but concentrate predominantly on the 5 leading viral vectors used in cancer gene therapy, specifically retroviruses, adeno-associated viruses, herpes viruses and lentiviruses with the most detailed analysis reserved for adenoviruses. The 3 main strategies for gene therapy in osteosarcoma are next summarized. As part of this review, the several prodrug-converting enzymes utilized in OS suicide gene therapy are examined. The text then turns to a discussion of adenovirus-mediated gene transfer and the need for tumor targeting via transductional or transcriptional approaches. Because of practical problems with use of replication-incompetent viruses in achieving complete tumor kill in vivo, virotherapy utilizing replication competent viruses has come to the fore. This topic is, thus, next reviewed which allows for a natural transition to a discussion of armed therapeutic viruses many of which are conditionally replicating adenoviruses carrying transgenes with established anti-tumor efficacy. We recognize that several other issues have arisen which hamper progress in the field of cancer gene therapy. We, therefore, review viral-induced toxicity in the host and vector delivery issues which have been found to potentially influence safety. We end with a brief perspective including commenting on animal models used in examining delivery strategies for osteosarcoma gene therapy. The challenges remaining are touched upon most especially the need to deal with pulmonary metastatic disease from OS.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

Effect of plasminogen activator (urokinase), plasmin, and thrombin on glycoprotein and collagenous components of basement membrane.

Tumor cells traverse basement membranes (BM) during the stages of the metastatic process. Penetration of the BM may involve proteolysis by enzymes directly or indirectly associated with tumor cells. This study evaluated the role of the serine proteases urokinase (plasminogen activator), plasmin, and another regulatory protease, alpha-thrombin, in the degradation of the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole human amnion BM. The BM components consisted of acid-extracted type IV collagen, pepsin fragments of collagen type IV, laminin, and fibronectin. Collagen type V (alpha A alpha B) associated with the peri-BM zone was also studied. The purity of the enzymes was verified by gel electrophoresis and inhibitor studies. Digestion of the BM components was performed at 25 degrees using matched activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagade fibronectin or any of the other BM components. Under the same 25 degrees (native) conditions, plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen, or alpha A alpha B (type V) collagen. alpha-Thrombn selectively degraded only the m.w. 400,000 chain of laminin, whereas plasmin degraded both the laminin chains. Digestion of laminin by the serine proteases was time and concentration dependent, as verified by a new degradation assay using [14C]laminin. A variety of normal and neoplastic cells were tested for the presence of laminin-degrading proteases. macrophages, polymorphonuclear leukocytes, and metastatic tumor cells contained a significant laminin-degarding activity. The activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by thrombin and plasmin at 35 degrees but not at temperatures below 33 degrees. Following treatment of whole-amnion BM with any of these enzymes, electron microscopy demonstrated preservation of the lamina densa. Immunohistology studies indicated that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment. The results suggest that these BM components are poor substrates for plasminogen activators and that plasmin alone is not sufficient to completely degrade the whole BM...

Flow cytometric analysis of human uterine sarcomas and cell lines.

Flow cytometric techniques were used to characterize multiple human uterine sarcomas and cell lines derived from some of these tumors. Analysis of DNA content showed that 9 of the 11 uterine sarcomas investigated were composed of at least one aneuploid population as well as a distinct diploid population. These data indicate that aneuploidy, as measured by flow cytometry, is a characteristic more common to uterine sarcomas than that previously reported for uterine adenocarcinomas. Unlike the original tumors, the cell lines established from three of the sarcomas contained predominantly diploid populations with only minor aneuploid populations. Treatment of one of the sarcoma cultures with tumor promoters did not result in an increase in the aneuploid populations. Tumors which arose in nude mice upon transplantation of two of the sarcomas did not contain the same distribution of tumor subpopulations as found in the original sarcomas. Apparently, the in vitro culture and and in vivo nude mouse conditions were not appropriate for maintaining the original equilibrium between the aneuploid and diploid subpopulations but instead provided a selective environment that resulted in the preferential growth of only certain tumor populations. Dual-parameter analysis of DNA content and alkaline phosphatase levels of one of the sarcomas were useful for distinguishing the aneuploid from the diploid population coexisting in this tumor. Our data suggest that flow cytometry is a valuable tool to analyze the characteristics of the tumor populations residing in primary uterine sarcomas as well as to determine which of these tumor subpopulations survive in culture and transplantation to nude mice.

Targeted gene delivery to Kaposi's sarcoma cells via the fibroblast growth factor receptor.

Kaposi's sarcoma (KS) is a major AIDS-related malignancy associated with significant morbidity and mortality. Current chemotherapeutic regimens are associated with a dismal prognosis. In an effort to develop a new approach to KS treatment, we devised a gene therapy-based adenovirus retargeting schema that redirects the adenovirus to fibroblast growth factor receptors endogenously present on the cell surface of KS cells. By using a bifunctional conjugate consisting of a blocking antiadenoviral knob Fab linked to basic fibroblast growth factor, FGF2, the gene transduction of KS cells was enhanced 7.7-44 fold; recombinant adenoviruses encoding either the firefly luciferase reporter gene, or the herpes simplex thymidine kinase gene, demonstrated quantitative enhancement of expression in the KS cell lines. In this regard, two KS cell lines that were previously refractory to native adenovirus transduction could be successfully transduced by the addition of the conjugate. This study thus addresses the utility of adenoviral retargeting to the FGF receptor in KS cells that are ordinarily transduction refractory to standardized approaches and allows practical development of gene therapy approaches for the treatment of human KS.

Intracellular expression of a single-chain antibody directed against human papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects.

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.