Faculty Bibliography

Our aim was to identify conditions which would permit the development of spontaneous metastasis of a human tumor in nude mice in a rapid and predictable manner and to explore ways to quantitate metastasis. Using a human squamous carcinoma--HEp3--we determined that invasiveness and metastasis were influenced by the host. HEp3 cells grew very rapidly and without a significant lag period in Balb/c and NIH(S)-II nude mice kept in aseptic conditions; a much longer lag period was observed in NIH-Swiss mice kept in conventional conditions. The HEp3 tumor displayed a highly invasive behavior in N-NIH(S)-II mice, in which it invaded the body wall, gaining access to the peritoneal cavity. Microinvasion was noted in all strains of mice inoculated with HEp3 cells. To prolong survival of the mice until metastases became evident, primary tumors were excised when they weighed 1-2 gm. N-NIH(S)-II and Balb/c nude mice, maintained in germ-free conditions, were most receptive to the development of metastases-lung metastases developed in 80% of these mice. Over 60% of all metastases were present within 4 weeks following the removal of the primary. Only 26% of tumor bearing NIH-Swiss developed lung metastases. Lung metastases were observed in some mice in the absence of local microinvasion, local tumor recurrence, and regardless of the presence of lymph node involvement. They were also noted in mice from which primary tumors were not excised. We compared three methods of lung metastasis detection: histology, detection of tumor cells in the cultures of lung minces, and the measurement of the levels of human urokinase-type plasminogen activator directly in the lysates of lungs. The detection of tumor cells in cultures of lung minces appeared to be the most sensitive of these methods and the determination of enzyme in lung lysates seemed to hold most promise for a quantitative approach. These data indicate that, the type of tumor, as well as the genetic background and the maintenance conditions of the host, have to be carefully selected to ensure the successful outcome of the particular tumor-host interaction being studied. Adherence to these guidelines allowed us, in the case of the HEp3 tumor, to develop a rapid, predictable, and efficient model in which to study factors affecting metastasis of this human tumor

Agents that slow cellular proliferation usually stimulate myeloid differentiation. The demonstration in this report of an anomalous inhibitory behavior of the epipodophyllotoxin VP16-213, an agent known to inhibit the enzyme DNA topoisomerase II, prompted us to investigate the role of this enzyme in both changes in DNA supercoiling and in DNA strand breakage and reunion events occurring during the induction of neutrophil-granulocyte differentiation. We recently reported that retinoic acid, an inducer of granulocytic differentiation, stimulates transient relaxation of DNA supercoiling. We now show that this is associated with the formation of small numbers of protein-linked DNA breaks (a characteristic of topoisomerase reactions). Both events are perturbed by VP16-213, and since this agent inhibits subsequent differentiation, these observations raise the possibility of a role for DNA topoisomerase II in granulocytic differentiation. The possible relevance of these findings to mechanisms of leukemogenesis is discussed

Previous studies have shown that the response of patients with acute myeloid leukemia to induction chemotherapy can be predicted by the species of plasminogen activator that their cells secrete. Patients whose cells secreted tissue plasminogen activator (tPA) only failed to respond to combination chemotherapy. Individuals whose leukemic cells display features of the early progenitor phenotype also respond poorly to therapy. This suggested that the two species of plasminogen activator secreted by leukemic cells might be produced by normal cells at distinct stages of differentiation. These results indicate that the secretion of the two enzyme types is a differentiation-linked property of normal cells with tPA being produced by granulocyte/macrophage progenitors and urokinase by more differentiated cells and by mature neutrophils and macrophages

Permanent cell lines (UCT-Mel 1 through 7) were established from biopsies of metastatic tissue taken from seven patients with malignant melanoma. Cells from these lines were all aneuploid and all grew as non-contact-inhibited, adherent monolayers. All of the lines, with the remarkable exception of UCT-Mel 6, formed tumours in nude mice, expressed the melanoma M-18 antigen and synthesized plasminogen activators exclusively of the tissue-type. UCT-Mel 6 cells were non tumourigenic, they lacked the M-18 antigen and they synthesized plasminogen activators exclusively of the urokinase type. UCT-Mel 1 and UCT-Mel 2 formed pigment in vitro and both of these lines showed an increase in pigment content and tyrosinase synthesis with increasing cell density. The rate of plasminogen activator released by UCT-Mel 1 and UCT-Mel 3 declined strikingly as the cells became confluent. Assuming that proteolytic activity is required for cell migration in vivo; that tyrosinase synthesis reflects expression of the differentiated phenotype and that melanoma cells retain some of the characteristics of neural crest cells, we suggest that the effects of confluence and close cell-cell contact provide a useful experimental counterpart for the study of normal neural crest all behaviour that is characterized by an inverse relationship between migration and a protease secretion on the one hand and pigmentation on the other

We have studied the regulation by glucocorticoids and dibutyryl cAMP of the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and a Mr approximately 54000 plasminogen activator inhibitor accumulated in serum-free conditioned culture fluid by a human fibrosarcoma, a human glioblastoma and a human melanoma cell line (HT-1080, UCT/gl-1 and Bowes). For the quantitation of u-PA and t-PA, we used sandwich-type ELISA with a combination of polyclonal and monoclonal antibodies. For an estimation of variations in the amount of the inhibitor, we used sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by Coomassie blue staining of conditioned culture fluid proteins, the inhibitor protein band being identified by its selective removal by passage of the conditioned culture fluids through a column with monoclonal antibodies against the inhibitor. The modulation of the 3 proteins by the hormonal agents varied greatly between the cell lines. The proteins were independently regulated, in the sense that the hormonal agents did not concomitantly change their levels in the direction expected either to increase or decrease total extracellular plasminogen activator activity. In conditioned culture fluids containing both t-PA and inhibitor, the two were present in the medium as a Mr approximately 120 000 complex. In contrast, no u-PA inhibitor complexes were found in conditioned culture fluid from any of the cell lines; this is likely to be due to the occurrence of u-PA in the culture fluid in the one-chain proenzyme form, which, unlike active u-PA, does not react with the inhibitor. These findings illustrate the complexity of the regulation of extracellular plasminogen activator activity, and imply that the presumed functional diversity of u-PA and t-PA may be related to their independent regulation

We have found that live and ethanol-fixed fibroblasts, when covered with conditioned medium containing tissue plasminogen activator, associate with the enzyme and remove it from the medium. Binding of tissue plasminogen activator to fixed cells showed equilibrium kinetics with maximal uptake corresponding to 2.4 units of enzyme per 10(6) fixed cells. Enzyme bound to fixed cells could activate plasminogen and produce plaques of caseinolysis in casein-plasminogen-agar overlays. Electrophoretic analysis showed it covalently attached to a fibroblast component with a molecular weight of 40,000-50,000. Sequestration of tissue plasminogen activator by live fibroblasts showed nonsaturable first order kinetics with a rate constant of 0.465/hr. We conclude that active enzyme is bound to a surface receptor, then internalized and degraded. Fibroblasts did not release the binding molecule into the medium; binding of tissue plasminogen activator from the medium was unaffected by heparin or thrombin. This phenomenon differs from that described by Baker et al. and ascribed to 'proteasenexin.'

This investigation was undertaken to examine the extent to which leukemic cell functions are susceptible to regulation in vitro and to investigate their heterogeneity in this regard. Since plasminogen activator release is known to be a modulatable cellular function that can be influenced by antiinflammatory steroids and tetradecanoyl phorbol acetate (TPA), the effect of these two compounds on the secretion of urokinase- or tissue-type enzymes by leukemic cells was studied. The release of both enzyme species could be stimulated or suppressed by these substances by mechanisms that were inhibitable by actinomycin-D and hence required transcription of new mRNA. Plasminogen activator release by cells from 41/45 patients with AML was either stimulated or inhibited by 10(-7) M dexamethasone, implying that most AML cells possess glucocorticoid receptors. In 26/45 cases, the enzyme was inhibited by this steroid to less than 25% of control values. Pronounced inhibition of this degree was not encountered with normal polymorphonuclear leukocytes. Plasminogen activator secretion by AML cells was profoundly inhibited in 20/41 cases by 1 ng/ml TPA and stimulated in 8/41 cases. Leukemic blasts varied considerably in their response to dexamethasone and TPA. Plasminogen activator release should prove a sensitive means of monitoring the responses of AML cells to biologically active compounds

Peripheral blood cell preparation from 23 normal subjects and 72 patients with acute and 32 patients with chronic myeloid leukemia were cultured in vitro and released plasminogen activators were analyzed. The quantity of plasminogen activator secreted by leukemic cells varied widely and could not be correlated with the clinical severity of the disease. Immunochemical and electrophoretic techniques have been used to show that normal peripheral blood granulocytes released exclusively urokinase-like plasminogen activator, whereas leukemic cells secreted either urokinase or a tissue activator-like enzyme. The molecular species of enzyme released by acute myeloid leukemic cells may serve as a diagnostic marker of relevance to the management of this disease, since patients with acute myeloid leukemia whose cells released only tissue plasminogen activator did not respond to combination chemotherapy. Tissue plasminogen activators released by leukemic cells may display an unusual electrophoretic pattern that resembles that shown by urokinase. Immunochemical procedures are therefore essential for the correct identification of these enzymes