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Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.

Routine diagnosis of pathology images transmitted over telecommunications lines remains an elusive goal. Part of the resistance stems from the difficulty of enabling image selection by the remote pathologist. To address this problem, a telepathology microscope system (TelePath, TeleMedicine Solutions, Birmingham, Ala) that has features associated with static and dynamic imaging systems was constructed. Features of the system include near real time image transmission, provision of a tiled overview image, free choice of any fields at any desired optical magnification, and automated tracking of the pathologist's image selection. All commands and images are discrete, avoiding many inherent problems of full motion video and continuous remote control. A set of 64 slides was reviewed by 3 pathologists in a simulated frozen section environment. Each pathologist provided diagnoses for all 64 slides, as well as qualitative information about the system. Thirty-one of 192 diagnoses disagreed with the reference diagnosis that had been reached before the trial began. Qf the 31, 13 were deferrals and 12 were diagnoses of cases that had a deferral as the reference diagnosis. In 6 cases, the diagnosis disagreed with the reference diagnosis yielding an overall accuracy of 96.9%. Confidence levels in the diagnoses were high. This trial suggests that this system provides high-quality anatomic pathology services, including intraoperative diagnoses, over telecommunications lines.

Originally conceived and applied for the treatment of inherited monogenetic defects such as adenosine deaminase deficiency and cystic fibrosis, gene therapy was later applied to the treatment of cancer. Such a genetic strategy seemed rational given the recognition that cancer typically develops in a multistep process involving alterations of a number of different genes as demonstrated in familial polyposis and colorectal cancer through the work of Vogelstein et al. Because of the numerous alterations that may result in the eventual development of cancer, there is no obvious single choice for a therapeutic gene. Although one may view this as an obstacle, it also allows for a variety of possible therapeutic interventions. This review focuses on the known genetic defects that occur in ovarian cancer, the gene therapy strategies suggested by such defects, and the approaches under current development for the treatment of this disease. As such, this work also describes some of the approved human gene therapy protocols. Finally, an overview of the problems and directions for future growth and research is presented.

We have previously demonstrated that an intracellular antibody (sFv) directed against erbB2 can achieve a specific cytotoxicity in erbB2 overexpressing cancer cells of varying histogenesis. In order to further delineate the mechanistic basis of the induced apoptosis, transient and stable cotransfections were performed. Transient cotransfection of erbB2 mutant and chimeric molecules demonstrated that the cytoplasmic domain of erbB2, or the homologous cytoplasmic domain of the epidermal growth factor receptor, is required for apoptosis induction. These results were confirmed in assays utilizing differential derivation of stable clones. To examine the effects of varying ratios of the anti-erbB2 sFv and its target erbB2 we performed additional cotransfection experiments in erbB2 negative target cells. When erbB2 levels are held constant, observed cytotoxicity is proportional to the amount of sFv added. In addition, when sFv levels are held constant, increasing levels of cotransfected erbB2 can overcome the apoptotic response. These results indicate that a minimal threshold level of the sFv and its target are required to induce cytotoxicity. To examine this phenomenon in an erbB2 positive cell line, SKOV3 ovarian carcinoma cells were utilized to derive a stable clone expressing low levels of sFv. When this cell line was compared to the parental SKOV3 cell line, it was shown that less exogenous sFv was needed to induce cytotoxicity in the clone already expressing low levels of sFv, indicating that endogenous and exogenous levels of sFv are additive. In summary, the results presented here indicate that the carboxy-terminus of the intracellular domain of the erbB2 molecule is involved in the induction of apoptosis. Furthermore, the expression levels of the sFv and its target protein need to overcome a threshold level in order to achieve a cytotoxic response.

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.

The delineation of the molecular basis of cancer in general, and of ovarian carcinoma in particular, allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To this end, three main approaches have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. For each of these conceptual approaches, human clinical protocols, including those specific for ovarian carcinoma, have entered phase I clinical trials to assess dose escalation, safety, and toxicity issues. However, major problems remain to be solved before these approaches can become effective and commonplace strategies for the treatment of cancer. In this regard, an examination of the applications of gene therapy for ovarian carcinoma can exemplify the rationality and the problems observed in the development of gene therapy, and may illustrate prospects for their solution that are being refined, including current efforts in our laboratory. An overriding obstacle is the basic ability to deliver therapeutic genes quantitatively, and specifically, into tumor cells. As vector technology fulfills these requirements, it is anticipated that promising results already observed in preclinical studies will translate quickly into the clinical setting for amelioration of this life-threatening disease in women.

Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.

1 alpha,25-Dihydroxyvitamin D (1,25 D; also know as calcitriol), the hormonal form of vitamin D, can inhibit the proliferation and promote the differentiation of human prostate adenocarcinoma cells. However, little is known about the effects of 1,25 D on the invasive ability of prostate cancer cells. We used an in vitro bioassay of cell invasion (Amgel assay) to examine the effects of 1,25 D and a "noncalcemic" vitamin D analogue, 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (16-23-D3), on the invasiveness of three well-characterized human prostate carcinoma cell lines: DU 145, PC-3, and LNCaP. PC-3 and LNCaP cells were poorly invasive in Amgel and were hardly affected by treatment with 1,25 D or 16-23-D3 (< 3%). Conversely, DU 145 cells were highly invasive in Amgel, and their invasion was markedly inhibited by 1,25 D and 16-23-D3 (maximally 66 and 59.4% respectively). This effect was both dose-dependent, with maximal inhibition at 1 x 10(-7) M and 72 h. Significant inhibition of invasion was observed at physiological levels of 1,25 D. Neither proliferative indices nor cell cycle kinetics were altered during the experimental exposure. Treatment with 1,25 D and 16-23-D3 caused a selective decrease in the secreted levels of type IV collagenases (MMP-2 and MMP-9). These findings support the hypothesis that 1,25 D reduces the risk of invasive prostate cancer and suggest a role for vitamin D compounds in the chemoprevention of invasive prostate cancer.

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.

We have previously shown that adenoviral-mediated delivery of an anti-erbB-2 intracellular single-chain antibody (sFv) causes specific cytotoxicy in erbB-2-overexpressing ovarian carcinoma cells. Furthermore, intraperitoneal delivery of the anti-erbB-2 sFv enhances survival and reduces tumor burden in a xenograft model of human ovarian carcinoma in SCID mice. These findings have led to an RAC-approved Phase I clinical trial for patients with ovarian cancer. In this report, we show that expression of the anti-erbB-2 sFv could be readily detected in target tumor cells by in situ hybridization methodology. PCR analysis of DNA extracted from various murine tissues demonstrated that the anti-erbB-2 sFv remained localized to the peritoneum. Delivery of the sFv to the non-erbB-2-overexpressing REN mesothelial and Hep G2 hepatocellular carcinoma cell lines was not deleterious to either one, affirming the tumor specificity of this gene therapy strategy. In addition, histopathological analysis of various tissues showed that adenoviral-mediated delivery of the anti-erbB-2 sFv to immunocompetent mice with either primary exposure or previous vector challenge at different doses produced no abnormal changes when compared to untreated animals. These findings suggest that adenoviral-mediated delivery of the anti-erbB-2 sFv in a human context can be effectively assayed, is potentially free of vector-associated toxicity, and retains biologic utility based on tumor specificity.

erbB-2 is a cell surface transmembrane glycoprotein which, when overexpressed, has been shown to be relevant to intrinsic tumor cell chemoresistance. Thus, strategies to down-regulate cell surface erbB-2 have resulted in enhanced tumor cell chemosensitivity. We have recently reported a gene therapy strategy to down-modulate erbB-2 expression using a plasmid construct encoding an intracellular single chain antibody. Therefore, we now demonstrate enhanced chemosensitivity to cis-diamminedichloroplatinum in erbB-2 overexpressing tumor cells and a model system of stable clones using an intracellular single chain antibody. These findings are consistent with the hypothesis that erbB-2 plays a role in tumor cell chemoresistance. In addition, these findings represent a novel gene therapy approach to overcome erbB-2-mediated tumor cell chemoresistance.

Cell adhesion to and migration through extracellular matrices (ECM) are critical events in tumor invasion and metastasis. Previous work by us had demonstrated that signaling of epidermal growth factor receptor (EGFR) confers an oncogenic phenotype on NR6 cells and that these cells when transfected with holo EGFR demonstrate greater motility and invasiveness than cells carrying a carboxy-terminal truncated EGFR. Recently, a cell surface glycoprotein, CD44, has been implicated in cell-ECM adhesion involved in tumor cell migration, signal transduction, and metastasis. We investigated whether EGF regulates cellular interactions with ECM components, and in particular, hyaluronate, by modulating CD44 expression. In vitro cell attachment assays on hyaluronate-coated plates demonstrated similar basal level of binding (approximately 33%) for murine NR6 parental cells devoid of endogenous EGFR (P) or expressing wild-type EGFR (WT), while a time-dependent increase in binding was observed in WT cells stimulated with EGF. Additionally, utilizing monoclonal antibody blocking assays, CD44, but not EGFR, was shown to be directly involved in this attachment. Both WT and P cells possessed equivalent 95 kDa bands on immunoblots, corresponding to CD44. The existence of CD44 mRNA was verified by RT-PCR using synthetic oligonucleotides in which a 1.1 kb cDNA was detected in both cell lines and confirmed by DNA sequencing. After 24-h exposure to exogenous EGF, an increase in CD44 protein and mRNA expression was found in WT cells, but not in P cells, supporting the contention that a functional EGFR signaling pathway is required for CD44 regulation. Thus, EGF stimulates cell binding to hyaluronate in vitro by regulating CD44 expression.

Methods were developed to achieve targeted eradication of the erbB-2 oncoprotein using gene constructs encoding anti-erbB-2 intracellular single-chain antibodies. This method of genetic intervention caused a marked cytocidal effect in erbB-2-overexpressing human ovarian tumor cells. Evaluation of the mechanistic basis of this phenomenon demonstrated that programmed cell death had been induced. Significantly, no cytocidal effect was observed in non-erbB-2-overexpressing tumors. The induction of apoptosis could be shown to be secondary to the intracellular antibody-mediated ectopic localization of the erbB-2 oncoprotein. Thus, the strategy of selective oncogene "knock-out" using intracellular antibodies represents a novel anticancer gene therapy strategy that offers the potential to achieve highly specific, targeted eradication of human tumor cells.

AA-amyloid has been produced experimentally in animal models, allowing the study of mechanisms involved in AA-amyloidogenesis, but those involved in renal AL-amyloidogenesis have not been adequately investigated due, in part, to lack of appropriate in vitro models. Rat and human mesangial cells were grown on a human extracellular matrix (Amgel) derived from normal tissues and on coverslips in the presence of 10 microliters of amyloid enhancing factor (AEF) per milliliter of media and 10 micrograms/ml monoclonal lambda light chains (LCs) obtained from two patients with AL-amyloidosis. Two additional lambda LCs derived from the urine of patients with myeloma and tubulointerstitial renal disease were used as controls. To verify amyloid deposition, light and electron microscopic examination, as well as Congo red and thioflavin T staining, were performed on samples incubated under different experimental conditions. Intracellular and extracellular amyloid was identified in samples incubated for 24 hours with human mesangial cells (for 48 hours with rat mesangial cells), amyloidogenic monoclonal LCs, and AEF. The amount of amyloid detected, which increased with longer incubation times, was found to be most abundant at 14 days. Amyloid was not present in cultures of mesangial cells incubated with amyloidogenic LCs alone or in the absence of mesangial cells. Likewise, incubation of mesangial cells with amyloidogenic LC or AEF separately or amyloidogenic LC in the presence of AEF but without mesangial cells did not result in amyloid formation. Amyloid was not seen when LCs obtained from the urine of patients with tubulointerstitial renal disease were incubated with AEF and mesangial cells. AL-amyloid production requires all three components--mesangial cells, amyloidogenic LCs, and AEF. In addition, amyloid was detected intracellular in mesangial cells, supporting the hypothesis that the production of AL-amyloid in the kidney requires intracellular processing by these cells. This system provides a unique experimental model to study renal AL-amyloidogenesis and a platform to explore mesangial cell-matrix interactions.

Tarsal tunnel syndrome is a compressive neuropathy caused by intrinsic or extrinsic pressure on the posterior tibial nerve or one of its terminal branches. A mass in association with tarsal tunnel syndrome is most likely a benign tumor or tumor-like condition, although a more malignant tumor must be in the differential diagnosis. We report an unusual case of an extraskeletal osteosarcoma causing tarsal tunnel syndrome.

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.

Prostate carcinomas often present an autocrine stimulatory loop in which the transformed cells both express the EGF receptor (EGFR) and produce activating ligands (TGF alpha and EGF forms). Up-regulated EGFR signalling has been correlated with tumor progression in other human neoplasia; however, the cell behaviour which is promoted remains undefined. To determine whether an EGFR-induced response contributes to cell invasiveness, we transduced DU-145 human prostate carcinoma cells with either a full-length (WT) or a mitogenically-active but motility-deficient truncated (c'973) EGFR. The DU-145 Parental and two transgene sublines all produced EGFR and TGF alpha, but the transduced WT and c'973 EGFR underwent autocrine downregulation to a lesser degree, with more receptor remaining intact. DU-145 cells transduced with WT EGFR transmigrated a human amniotic basement membrane matrix (Amgel) to a greater extent than did Parental DU-145 cells (175 +/- 22%). Cells expressing the c'973 EGFR invaded through the Amgel only to about two thirds the extent of the Parental cells (62 +/- 23%). A monoclonal antibody which prevents ligand-induced activation of EGFR decreased the invasiveness of WT-expressing cells by half and Parental cells by a fifth, but had little effect on the invasiveness of c'973-expressing cells; with the result that in the presence of antibody, all three cell lines transmigrated the Amgel to the same extent. The different levels of invasiveness between the three sublines were independent of cell proliferation. These findings demonstrated that EGFR-mediated signals increase tumor cell invasiveness and suggested that domains in the carboxy-terminus are required to signal invasiveness. As an initial investigation into the mechanisms underlying the EGFR-mediated enhanced invasiveness, we determined whether these cells presented different collagenolytic activity, as the major constituents of Amgel are collagen types I and IV. All three sublines secreted easily detectable levels of gelatin-directed proteases and TIMP-1, with WT cells secreting equivalent or lower levels of proteases. The proteolytic balance in these cells did not correlate with invasiveness. These data suggest that the TGF alpha-EGFR autocrine loop promotes invasiveness and that this is accomplished by signalling cell properties other than differential secretion of collagenolytic activity.

Objective: Overexpression of the tyrosine kinase receptor erbB-2 is important in the pathogenesis of a variety of neoplasms including ovarian cancer. As a strategy to selectively eradicate erbB-2-overexpressing tumor cells, an anti-erbB-2 single-chain immunoglobin (sFv) gene was constructed to direct expression of intracellular anti-erbB-2 antibody. The purpose of this study is to establish the antitumorigenicity of this strategy in in vitro and in vivo models., Methods: An anti-erbB-2 sFv construct containing an endoplasmic reticulum (ER)-directed leader sequence was transiently expressed in the human ovarian carcinoma cell line SKOV3 using the adenovirus-polylysine vector. SKOV3 cells transfected with a non-ER form of an anti-erbB-2 sFv construct or an irrelevant plasmid DNA served as controls. Antitumorigenicity, as measured by anchorage-independent growth in soft agar and subcutaneous (sc) tumor formation, was analyzed. The ability to achieve a biological effect with relevant sFv in murine orthotopic xenograft models and in primary human ovarian cancer cells was also evaluated., Results: The ER form of anti-erbB-2 sFv was found to exert a marked antineoplastic effect on the SKOV3 cell line resulting in an arrest of anchorage-independent growth. A significant increase in sc tumor volume was noted in animals challenged with control constructs. In marked contrast, complete tumor eradication was noted at necropsy 80 days after sc transplantation in the group challenged with the ER-directed anti-erbB-2 sFv gene. Intraperitoneal treatment of malignant ascites in human tumor xenograft models with the ER form of the anti-erbB-2 sFv gene resulted in profound downregulation of cell surface erbB-2 in retrieved ovarian cancer cells. The ER-directed anti-erbB-2 sFv also elicited a significant cytotoxic effect in transfected primary ovarian cancer cells obtained from a patient with malignant ascites., Conclusion: The ability to selectively "knock out" erbB-2 demonstrates that this strategy can induce a significant antineoplastic effect in ovarian cancer cells overexpressing this growth factor receptor. In addition, the ability to accomplish selective abrogation of erbB-2 expression in animal treatment models and to transfect and eradicate primary ovarian cancer cells justifies further investigation of this novel strategy in ovarian cancer patients.

Transitional cell carcinoma (TCC) of the bladder is associated with characterized lesions in dominant and recessive oncogenes. The understanding of the molecular basis of tumorigenesis in these instances makes possible the application of gene therapy strategies for TCC. In this regard, the ability to directly access the epithelium of the genitourinary (GU) tract via the urethra provides a practical means to implement these various gene therapy approaches. We thus explored vector strategies to accomplish direct in vivo transduction of GU epithelium. Initially, three human (HT 1197, HT 1376, T24) and one mouse (MBT-2) TCC cell lines were transduced using a recombinant adenoviral vector expressing the firefly luciferase reporter gene, rAd-CMV-Luc. In these studies, reporter gene expression was found to be significantly elevated above background for all four cell lines. Of note, the TCC cell lines HT 1197 and HT 1376 showed expression levels comparable with the cervical carcinoma cell line HeLa, a cell line previously shown to be highly susceptible to recombinant adenovirus-mediated gene transduction. An in vitro time course for T24 and MBT-2 using rAd-CMV-Luc showed peak expression 1 day after transduction for the T24 line and 3 days after transduction for the MBT-2 line, with detectable levels of expression persisting for at least 7 days. As a next step, human and mouse primary tissue deriving from the GU epithelium were transduced using rAd-CMV-Luc. In this assay, luciferase expression levels significantly above background were observed in both instances.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors have analyzed several endocrine and metabolic parameters in polyoma large T transgenic mice (PyLT-1) that develop adrenocorticotropic hormone (ACTH)-immunoreactive pituitary tumors and in nontransgenic mice with tumor transplants. All clinically ill PyLT-1 mice (13 to 16 months of age) had pituitary macroadenomas and elevated plasma ACTH levels. Compared to PyLT-1 transgenic mice, the ACTH plasma concentrations in immunocompetent mice with transplant tumors were markedly raised. In these animals, a secondary effect of hypercorticotropism was documented by a moderate hyperglycemia. Furthermore, mice with transplant tumors had a pathological weight increase from the time the tumor was palpable. The present study supports and extends the authors' previous morphological documentation of the similarity between the ACTH-producing tumors in this mouse model and human Cushing's disease.

Interactions between the extracellular matrix (ECM) and the neoplastic cells they envelop are thought to play a fundamental role in those cells' ability to invade, one of the key events in the metastatic cascade. Cellular transport of amino acids, in turn, is known to be mediated by functionally distinct membrane transport systems and is modulated by substrate bioavailability in the microenvironment. We postulate that certain advantages enjoyed by a neoplastic cell population over their normal counterparts (for example, increased proliferating capability and invasiveness across ECM barriers) are linked to changes in the cells' differential control of amino acid transport (aaT) via host ECM-tumor cell generated signals. Our studies suggest that active transport of neutral amino acids modulates a cells' functional behavior among phenotypically distinct human transformed cell types, irrespective of whether they are categorized as a sarcoma, melanoma, or carcinoma. We present preliminary laboratory evidence which has lead us to formulate a series of working hypotheses as follows: 1. aaT systems operating in both non-transformed and transformed human cells exhibit differential transport kinetics; 2. adaptive regulation of certain amino acids via cell-specific aaT systems alters a cell's ability to invade human ECM; and 3. aaT induction involves changes both at the cellular and molecular levels. This report, therefore, provides experimental support, and suggests a possible mechanism, to explain how neutral amino acids, acting as nutrient signalling factors (along with other biologic elements) within the cell milieu, have the capability of regulating the phenotypic nature of human neoplastic cells.

Increasing evidence indicates that cationic liposomes are capable of safely transferring foreign genes to pulmonary epithelium in vitro and in vivo. To transfer reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) to mammalian respiratory epithelium we used two cationic lipid formulations: N-[1-(2,3-dioleoyloxy)propyl] N,N,N-triethylammonium chloride (DOTMA), and 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) at a 1:1 molar ratio with dioleoyl phosphatidylethanolamine (DOPE). Lipid-DNA conjugates containing either CFTR or LacZ were instilled directly into the airways of Sprague-Dawley rats. Rats treated with LacZ cDNA in vivo demonstrated expression in 30-50% of the large and medium-sized airways, with some airways showing high efficiency gene transfer and expression (in the most proximal airways, 70-80% of surface epithelial cells were positive for expression of a nuclear targeted LacZ). While control and LacZ treated tracheas mounted in Ussing chambers showed minimal stimulation of transepithelial chloride (Cl)-currents by cAMP (suggesting low levels of endogenous rat CFTR activity), tracheas taken from animals receiving CFTR exhibited significant forskolin-stimulated currents at 72 h after gene transfer. Human CFTR gene expression was also detected by polymerase chain reaction (PCR) analysis of reverse transcribed lung RNA. These results, together with previous studies using lipid-mediated gene transfer in mice, help confirm the potential for cationic lipid-mediated gene transfer in the gene therapy of cystic fibrosis in humans.

Tumor growth and metastasis are affected by changes in membrane lipid composition, however, little is known regarding the role of specific fatty acids in these pathological events. We investigated the effects of the long-chain saturated fatty acids (LCSFA), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) on two key steps of metastasis: cell adhesion and invasion into extracellular matrix (ECM). Using a new 72-hour ECM (Amgel) invasion assay, we demonstrated that the exposure of highly invasive human fibrosarcoma HT-1080 cells to 0.3 mM stearate inhibited their ability to traverse Amgel by 59.4 +/- 8%. In contrast, treatment of tumor cells with 0.3 mM myristate or palmitate had no effect. Microscopic examination revealed a time-dependent inhibition of tumor cell adhesion to the Amgel in the stearate-treated group. Cell adhesion assays further showed a series of rapid morphological cellular changes, i.e. retraction of processes, cell rounding, and subsequent detachment in the presence of stearate. These morphological events were both dose- and time-dependent. Viability of LCSFA-treated cells exceeded 80%. This stearate inhibition of HT-1080 cell adhesion was also observed with two other invasive human tumor cell lines. Similar treatment of HT-1080 cell with the unsaturated long-chain fatty acid oleate (C18:1) did not alter tumor cell adhesiveness. In contrast, nontransformed human fibroblasts (Hs-68) were unaffected by stearate treatment. This inhibition of cell adhesion by stearate was determined to be dependent upon laminin-containing ECM. Pretreatment of HT-1080 cells with stearate dramatically abolished their capacity to attach to laminin but not to collagen type IV or fibronectin matrices. Immunofluorescent studies with anti-beta 1 integrin receptor and antivinculin antibodies demonstrated beta 1 subunit and vinculin colocalization to focal adhesions in untreated HT-1080 cells adherent to laminin, in contrast to stearate-treated tumor cells. Further, stearate-induced changes were shown to be functionally coupled to integrins as an anti-beta 1 antibody markedly diminishes the adhesive ability of tumor cells to laminin. These data demonstrate stearate inhibits tumor cell adhesion, and therefore invasion, via a mechanism involving a laminin integrin receptor.

Overexpression of the tyrosine kinase receptor erbB2 is important in the pathogenesis of a variety of neoplasms. Based on this concept, targeted anti-cancer strategies have been designed to selectively eradicate erbB2 overexpressing tumor cells. These strategies have employed either monoclonal antibodies or antibody toxin molecules with specificity for the cell surface erbB2 protein. As an alternative strategy, anti-erbB2 single-chain immunoglobulin (sFv) genes were constructed to direct expression of intracellular anti-erbB2 antibodies. Expression of an endoplasmic reticulum (ER) form of the anti-erbB2 sFv resulted in a profound down-regulation of cell surface erbB2 in an erbB2 overexpressing ovarian carcinoma cell line. In addition, expression of the intracellular antibody resulted in marked inhibition of tumor cell proliferation. Whereas stable transfectants expressing the anti-erbB2 sFv could be dervied from non-erbB2 overexpressing cancer cell lines, expression of the intracellular antibody was incompatible with long-term survival of the erbB2 overpressing tumor cells. The ability to selectively 'knock-out' erbB2 demonstrates that cell surface localization of erbB2 is essential to its ability to induce aberrant cellular proliferation in tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Transgenic mice carrying the polyoma (Py) early region gene develop both vascular and bone tumors that express the transgene (R. Wang and V. L. Bautch, J. Virol. 65:5174-5183, 1991). To determine the correlation between bone tumor formation and transgene expression, mice of two lines that showed differences in tumor pattern were analyzed., Experimental Design: Py DNA encoding the early region gene was inserted into B6D2F1 mouse embryos. Transgenic mice were sacrificed at appropriate times in development, and the histopathology of the skulls and genotype analyses were performed independently. Py transgene expression was assayed in individual skulls or small groups of skulls., Results: In mice of the Py-4 line, Py oncogene expression was detectable in skulls from 14.5 days of development. These mice had normal skulls at birth, and they developed lesions histologically resembling osteosarcoma synchronously 1 to 2 weeks after birth. Mice of the Py-3 line did not express detectable levels of transgene at 3 weeks of age, and they did not develop detectable skull pathology at this age. Less than 20% of adult Py-3 mice developed calvarial osteosarcoma-like lesions, and a similar number of these mice expressed the transgene in the skull., Conclusions: These data show that Py transgene expression in bone of the skull is differentially regulated in mice of the two lines, and they indicate that epigenetic changes may be sufficient to complement expression of the Py oncogene in neoplastic bone formation during development.

The extracellular matrix (ECM) is actively involved in the growth and maintenance of normal and neoplastic mammalian cells. It has been suggested that the growth-promoting factors sequestered within the matrix exclusively regulate the observed phenomena. We postulate, however, that ECM components alone, when derived from normal human tissues, and devoid of major growth factors, can modulate the growth of human tumor cells in vivo. In this submission we provide evidence that Amgel, an ECM created by us from nontumorigenic human placental tissues, can enhance or retard human cell growth in vivo, depending upon type, when placed in the subcutaneous tissue of athymic mice. Further, we provide evidence that these results differ from those obtained utilizing a tumor-derived ECM. Our findings suggest that specific tumor cell-matrix interactions are responsible for the observed results. To the best of our knowledge, this is the first report of a normal, human tissue-derived ECM both promoting and inhibiting selected human tumor cell growth in vivo. Thus, Amgel should prove useful in elucidating the underlying mechanisms of cell-matrix interactions during the growth and progression of human neoplasms.

Previous studies have demonstrated that some human endometrial carcinomas contain an activating point mutation in codon 12 of the Ki-ras protooncogene. To examine the hypothesis that this mutation may occur at an earlier stage of neoplastic progression in the endometrium, we analyzed 89 samples of premalignant endometrial hyperplasia and an additional 84 samples of endometrial carcinoma for point mutations of Ki-ras codon 12. Mutations were found in all three types of endometrial hyperplasia, simple, complex, and atypical, with no clear evidence of a differential distribution in any particular type. Furthermore, the overall incidence of Ki-ras mutations in the hyperplasia specimens (16%) was similar to the incidence detected in carcinomas (18%), indicating that ras mutation may represent an early event in a subset of endometrial carcinomas. When the tissue samples were segregated as to country of origin, the frequency of this mutation was approximately 2-fold higher in hyperplasia and carcinoma samples from Japan than from the United States, where the incidence, clinicopathological characteristics, and risk factors for endometrial carcinoma differ dramatically. There was no apparent correlation, however, between ras mutation and any pathological, histological, or clinical parameter examined, except survival. The presence of a ras mutation was inversely associated with death from disease, suggesting that this molecular feature may characterize a subset of endometrial carcinomas with a good prognosis.

Normal mesenchymal cells within developing embryonic organs and transformed stromal cells in organs undergoing spontaneous carcinogenesis have the capacity for normal or altered expression of the extracellular matrix glycoprotein tenascin (Tn). Mesenchymal cell constituents of normal adult organs show only a very limited tendency to deposit Tn in their extracellular matrix. In the present study, we investigated whether malignant human mesenchymal cells derived from uterine sarcomas or normal human endometrial stromal cells partially transformed via transfection with selected oncogenes have the capacity to produce and deposit Tn. We reached the following conclusions: (1) compared with normal endometrial tissues, uterine sarcomas show heterogeneous, but increased, immunoreactive staining patterns exclusively within the extracellular compartment, regardless of the histologic subtype of the tumor; (2) in vitro, all normal and transfected stromal cells and cell lines examined secreted Tn into the tissue culture medium; (3) this secretory ability was not translated into morphologic uniformity, since immunoreactivity detected by confocal laser scanning microscopy was observed in only selected cell populations; (4) also, the deposition and the incorporation of Tn depended upon the density of transfected cells, and (5) double-staining experiments revealed that Tn could always be localized in close proximity to fibronectin. In summary, the production of Tn is increased in most cases of human uterine sarcoma. The capacity of stromal cells to deposit Tn in a matrix-like structure in vitro, rather than increase production of Tn, is correlated with the degree of neoplastic progression.

Transgenic mice that developed adrenocorticotropic hormone (ACTH)-producing pituitary tumors were generated with the polyoma early region promotor linked to a cDNA encoding polyoma large T antigen (PyLT). Light microscopic examination of the pituitaries showed normal morphology at 4 months of age, either unremarkable morphology or microadenoma formation at 9 months of age, and up to 5 mm large adenomas in clinically ill transgenic mice at 13-16 months of age. At age 9 months, transgenic mice weighed significantly more than corresponding control mice, but they began wasting at approximately 1 year of age. The adrenal glands of these older PyLT-1 mice showed a weight increase and exhibited a medullary hyperplasia. Subcutaneous transplants of transgenic pituitary tumors to nontransgenic, immunocompetent mice resulted in tumors with a morphology and ACTH immunoreactivity similar to the primary tumor. The effects of hypercorticotropism were more enhanced and occurred with a shorter latency in the mice carrying transgene pituitary transplants than in the PyLT-1 transgenic mice themselves. Moreover, these transplanted mice showed a weight increase with an axial deposition pattern and hypertrophy of the adrenal cortex that resembled the findings in human Cushing's disease. Plasma ACTH levels were significantly increased in clinically ill transgenic mice and even higher levels were found in the transplant mice. Thus, both murine models should be useful for studying Cushing's disease.

The BDII/Han rat develops spontaneous endometrial adenocarcinoma, which appears virtually identical histologically to human endometrial adenocarcinoma. The incidence rate of cancer formation in the rat is 90% and the mean lifetime of the animals is 22 months. This animal model therefore, is useful in the study of molecular aspects of spontaneous transformation as well as mammalian neoplastic progression. In this study we address the in-situ expression of tenascin, an extracellular matrix glycoprotein, during normal cyclic growth, during development of proliferative states, and during malignant transformation of the endometrium. Trace amounts of immunocytochemically detectable tenascin were found in 10% of young BDII/Han rats with a normal estrus cycle. In these inbred animals no tenascin was detectable in uteri without neoplastic progressive alterations of the endometrium. Tenascin immunoreactivity first appeared during proliferation in one of three uteri with cystic glandular hyperplasia. Prominent tenascin expression was detectable in all adenomatous hyperplasia, but restricted to the stromal mesenchyme, that surrounded the glands. In all endometrial adenocarcinomas tested, essentially the entire extracellular space of the stromal mesenchyme was immunoreactive with anti-tenascin antibodies while the epithelial glands themselves were negative. This staining pattern was observed independent of the degree of tumor differentiation or extent of myometrial invasion. The tenascin staining pattern was not significantly altered in tumors transplanted into the soft tissues of the neck of female BDII/Han rats. From our studies we conclude that tenascin may be a marker for the early detection of proliferative endometrial states.(ABSTRACT TRUNCATED AT 250 WORDS)

The biological behavior of pleomorphic adenomas (mixed tumors) of salivary gland origin is complex. Tumors with benign histologic features may exhibit recurrence and locally aggressive behavior especially after incomplete excision. A small percentage of pleomorphic adenomas have obvious malignant components in epithelial or in both epithelial and mesenchymal components and can metastasize. There are also rare case reports which appear to document typical pleomorphic adenomas of salivary gland with histologically identical visceral and lymph node metastases. Recently myoepithelial cell proliferation has been identified as a possible predictor of aggressive clinical behavior in otherwise histologically benign pleomorphic adenomas. We report such a parotid gland lesion with local recurrence and retroperitoneal spread. DNA-flow cytometry of cells from the paraffin-embedded primary and metastasis showed similar aneuploid populations. Aneuploidy appeared to reflect the malignant potential of this particular pleomorphic adenoma and suggests that DNA-flow cytometry of salivary gland tumors may yield important prognostic information.

Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix.

To investigate the involvement of tenascin, an extracellular matrix glycoprotein, in epithelial growth and malignancy, its specific distribution pattern in the human uterus was examined immunohistochemically. During the proliferative phase of the menstrual cycle, this antigen was found as a sharp band around the endometrial glands. The immunoreactivity persisted until the early postovulatory phase of the menstrual cycle, but was not detectable in the glandular or stromal compartment during this later secretory stage, instead endometrial arterioles were immunostained. In marked contradistinction, when antibodies directed against tenascin were applied to sections of endometrial adenocarcinoma, almost the entire extracellular space stained, whereas the neoplastic cells themselves were nonreactive, whatever the degree of tumor differentiation. In precancerous proliferative lesions of the endometrium, tenascin's presence was variable. It was detectable around some superficial glands demonstrating cystic hyperplasia and around all deeply situated glands at the endometrial/myometrial interface. In cases of adenomatous hyperplasia, tenascin immunolocalized throughout the extracellular space of the stroma and the staining intensity was increased as the hyperplasia became more atypical. We therefore conclude that tenascin may be a stromal marker for epithelial proliferative states including those associated with malignancies of the endometrium.

Fourty-six bronchial carcinoids, twelve tumourlets and twenty areas of neuroendocrine cell dysplasia (NED) were immunohistochemically evaluated for various neuroendocrine markers, S-100 protein (S-100), myelin basic protein, intermediate filaments, actin, Leu-7 and several neurohormonal polypeptides. Eighteen of the bronchial carcinoids (39.1%) showed a biphasic cell pattern, with abundant stellate-shaped S-100 positive cells (SC). SC were not reactive for chromogranin A, myelin basic protein, cytokeratins, neurofilaments, glial fibrillary acidic protein or actin, and were only occasionally weakly positive for vimentin. SC were not detected in the tumourlets nor in the NED observed. For comparison a group of other neuroendocrine tumours (11 gastrointestinal carcinoids, 4 pheochromocytomas and 4 paragangliomas) were immunostained for S-100, chromogranin A and actin. SC similar to the ones detected in the bronchial carcinoids could be detected in appendiceal carcinoids, paragangliomas and in two out of four pheochromocytomas. Our present data are in keeping with a Schwannian/sustentacular nature of SC rather than that of a histiocytic or myoepithelial nature. We suggest that SC-rich bronchial carcinoids are biphasic tumours, which could be designed "paraganglioid" bronchial carcinoids. The relationship between SC-rich bronchial carcinoids and tumourlets/NED is a matter of further investigation: SC-rich bronchial carcinoids may either differentiate in a biphasic pattern during tumoural growth or may not be histogenetically related to tumourlets.

Stromal cells isolated from normal human endometrium were cotransfected in primary culture with pSVc-myc, a plasmid containing a truncated c-myc gene regulated by simian virus 40 promoter, and pRSV neo, a plasmid containing a neomycin resistance gene regulated by Rous sarcoma virus (RSV) promoter. These cells demonstrated properties of transformed cells in vitro, including altered morphology, focus formation, anchorage-independent growth, chromosomal alterations, and tumor formation in athymic mice. When these cells were treated subsequently with a direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, they demonstrated higher colony-forming efficiency in soft agar and reduced tumor latency. Cells transfected with pRSV neo alone exhibited some properties associated with neoplastic transformation, including altered morphology and formation of colonies in soft agar. It is presumed that in normal cells transfected with pRSV neo, RSV long terminal repeats activated cellular genes that normally regulate growth of human endometrial stromal cells.

Ten cases of malignant mesothelioma (MM), as diagnosed by clinical history and light and electron microscopy, were studied with polyclonal antibodies directed against the basement membrane-specific proteins, type IV collagen and laminin, as well as with monoclonal antibodies which recognize two epitopes of the laminin receptor (LR). All formalin-fixed, paraffin-embedded mesothelioma tissues examined demonstrated intracytoplasmic immunoreactivity for the basement membrane proteins. Extracellular staining was minimal, analogous to the staining reactions observed in adenocarcinomas of the breast and lung, which on light microscopy mimicked the morphologic appearance of MM. Similarly, LRs were identified on MM cells by intense positive staining. Immunoreactivity was also evident on nonneoplastic mesothelioma and adenocarcinoma cells but with greater heterogeneity and less intensity. It may be concluded from these results that (a) malignant mesotheliomas have the ability to synthesize components of the basement membrane; (b) enhanced attachment to extracellular matrix by MM would be anticipated as laminin receptors are present in large numbers on the surface of mesothelioma cells; (c) the reason for lack of intensiveness by MM cells remains speculative; type IV-specific collagenase may play a role in regulating this function in these tumor cells.

A 41-year-old man with recognized polyostotic fibrous dysplasia since late childhood developed fibroblastic osteogenic sarcoma in the left tibia. Four months after the initial diagnosis, an intramuscular myxoma was discovered in the left thigh. Twenty years previously he had been found to be heterozygous for hemoglobins JBaltimore and S. Malignant transformation in fibrous dysplasia is unusual and may be associated in some individuals with prior irradiation. Soft tissue myxomas associated with fibrous dysplasia are even rarer. To the best of the authors' knowledge the occurrence of both of these lesions in a patient with fibrous dysplasia has been reported only once before. Patients with both fibrous dysplasia and myxomas may be at greater risk for malignant transformation than are individuals with only one of these lesions. There is no well-recognized association between hemoglobinopathies and either fibrous dysplasia or bone tumors. It is therefore probable that the rare constellation of findings is in this patient a stochastic event.

Two patients, a mother and her son, had idiopathic pulmonary hemosiderosis. In the son, the onset was acute and he died of massive hemoptysis. The mother's disorder developed less acutely and the condition improved after temporary respiratory support and massive corticosteroid therapy. This is the first reported instance of idiopathic pulmonary hemosiderosis occurring in more than one member of the same family. The diagnosis of idiopathic pulmonary hemosiderosis depends on compatible clinical and pathologic data and on exclusion of secondary causes of intrapulmonary hemorrhage.

Review of current data from the Intergroup Ewing Sarcoma Study (IESS) shows that Ewing sarcoma is rare in bones of the hands and feet. The 12 patients from the IESS protocols with hand or foot Ewing sarcoma are comparable to those already reported in the literature. With the exception of lesions in the calcaneus, the prognosis for disease-free survival is excellent. The radiographic features of hand and foot Ewing sarcoma are generally those of classic Ewing sarcoma: permeation, soft-tissue mass, and, often, associated sclerotic reaction. However, with the exception of sclerosis, features suggesting bone reaction and slow tumor growth in these patients were distinctly uncommon compared with Ewing sarcoma in general. Those radiographic features suggesting aggressive behavior were slightly more common than in the general Ewing population. Even so, neither the radiographic appearance nor the greatest dimension of the radiographic lesion can be shown to contribute significantly to prognostication in these patients. Apparently location of the lesion is important, since in the reported cases in the literature and in this series, lesions of the calcaneus fared poorly. The importance of this set of patients therefore relates to awareness and early recognition of an unusual appearance and location of Ewing sarcoma.

A girl, aged 2 years, 11 months, who had pain and swelling of the proximal humerus, was found to have osteoblastic osteogenic sarcoma of the humerus. Among 937 consecutive patients with osteogenic sarcoma examined at the Mayo Clinic, she is the youngest child seen, as well as only the sixth child younger than 6 years of age in this series.

Of 127 patients with primary neoplasms of the scrotal wall 10 had a solitary leiomyoma arising from the tunica dartos. These benign asymptomatic lesions occur in middle-age white men and are often incorrectly diagnosed. Simple surgical excision is curative. The 11th case is described because of the malignant course that followed irradiation. A cell arising from single smooth muscle cells within the corium is proposed as the cell of origin.

Monoclonal antibodies (MoAb) that cross-react with the shared epitopes of S100 protein have been prepared from mouse hybridoma cell lines and partially characterized. Nine of these MoAb were applied to sections of formaldehyde-fixed paraffin-embedded human tissues that were stained by immunohistochemical techniques. Three of these MoAb give uniformly and reproducibly positive staining in appropriate cell types when stained by avidin-biotin methods. Three of the MoAb were judged to be negative, although some MoAb gave inappropriate staining patterns. The three remaining MoAb showed either great heterogeneity in their staining patterns or intensities, or gave a lesser degree of reproducibility in a given tissue or neoplasm. One of the MoAb designated 15E2E2 that belonged to the first group of reproducibly staining antibodies was used to stain a larger number of normal human tissues and neoplasms. The staining that was observed appeared to recapitulate that which was previously described for conventional S100 protein antibodies. Monoclonal antibodies may, therefore, have a role in selected cases where standard microscopy is equivocal for a specific tissue diagnosis, or where independent verification of the diagnosis would be beneficial.