Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.

Trans-membrane control of the receptors on normal and tumor cells. II. Surface changes associated with transformation and malignancy.

Epstein-Barr virus (EBV) can display different forms of latent infection in B-cell lines in vitro; however, the types of infection normally established by the virus in vivo remain largely unexplored. Here we have approached this question by analyzing the types of viral RNAs present in mononuclear cells freshly isolated from the blood of 14 infectious mononucleosis patients undergoing primary EBV infection and 6 long-term virus carriers. Reverse transcription-PCR amplifications were carried out with a panel of oligonucleotide primers and probes which specifically detect (i) the EBER1 RNA common to all forms of latency, (ii) transcripts either from the Cp and Wp promoters generating all six nuclear antigen (EBNA1, -2, -3A, -3B, -3C, -LP) mRNAs or from the Fp promoter generating a uniquely spliced EBNA1 mRNA, (iii) the latent membrane protein (LMP1 and 2A) mRNAs, and (iv) the BZLF1 mRNA, an immediate-early marker of lytic cycle. Viral transcription in infectious mononucleosis mononuclear cells (and in the B-cell-enriched fraction) regularly included the full spectrum of latent RNAs seen during EBV-induced B-cell growth transformation in vitro, i.e., EBER1, Cp/Wp-initiated EBNA mRNAs, and LMP1/LMP2 mRNAs, in the absence of lytic BZLF1 transcripts. In addition, transcripts with the splice pattern of Fp-initiated EBNA1 mRNA, hitherto seen only in vivo in certain EBV-positive tumors, were frequently detected. In long-term virus carriers, the mononuclear cells were again positive for latent (EBER1) and negative for lytic (BZLF1) markers; Cp/Wp-initiated RNAs were not detected in these samples, but in several individuals it was possible to amplify both Fp-initiated EBNA1 mRNA and LMP2A mRNA signals. We suggest (i) that primary infection is associated with a transient virus-driven expansion of the infected B-cell pool through a program of virus gene expression like that seen in in vitro-transformed cells and (ii) that long-term virus carriage is associated with a switch from Cp/Wp to Fp usage and thus to a more restricted form of latent protein expression that may render the infected cells less susceptible to recognition by the virus-specific cytotoxic T-cell response.

Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state.

The Epstein-Barr virus.

Variation of Epstein-Barr virus (EBV) in respect to its effect on the properties of transformed cells was probed. Human umbilical cord leukocytes from six different individuals were transformed in vitro by either B95-8 (B) or QIMR-WIL (Q) strains of EBV and subsequently 12 lymphoblastoid cell lines (six B-derived and six Q-derived lines) were established. The B lines and Q lines were different in the expression of EBV genome i.e. production of virus or viral antigens, and in other properties including growth pattern and immunoglobulin production. The most striking differences between the two groups lay in their capacity to produce infectious virus and in the shape of the cell-clumps. The results suggest that different strains of EBV may induce transformed cells with different characteristics.

Characteristics of cell lines derived from human leukocytes transformed by different strains of Epstein-Barr virus.

Identification of the target cells in human B lymphocytes for transformation by epstein-barr virus

The B95-8 strain of Epstein-Barr virus (EBV) induced colony formation of human umbilical cord-blood leukocytes in soft agar medium. One-hit response relationship between the number of colonies and the virus dose was observed with high dilutions of the virus preparation. However, there was a presumed cell-killing effect with low dilutions of virus. The colonies were similarly induced, but with a lower efficiency, in adult peripheral blood leukocyte cultures infected with the virus. The colony-forming activity of EBV was neutralized by anti-EBV-positive but not by negative human sera. The cells in colonies were capable of growing continuously and carried EBV-associated nuclear antigen. Thus, it was evident that the colony formation was caused by clonal transformation by EBV.

Clonal Transformation of Human Leukocytes by Epstein-Barr Virus in Soft Agar

EBNA-positive lymphoblast cells were detected in 0.1 to 0.9% of the T-cell-depleted lymphocytes obtained from peripheral blood samples of five patients with infectious mononucleosis (IM). The same blood specimens from four of the five patients contained cells that formed EBNA-positive colonies in soft agar containing EBV antibodies. The ratio of the colony formers to EBNA-positive cells was higher in blood samples taken early in the disease than in those obtained in later stages of the disease. The present results strongly suggest that EBV-transformed cells are present in the peripheral circulation of IM patients and that such cells can directly give rise to immortalized cell lines in vitro.

Simultaneous presence of EBNA-positive and colony-forming cells in peripheral blood of patients with infectious mononucleosis.

Critical evaluation of the anti-complement immunofluorescence (ACIF) test for Epstein-Barr virus-associated nuclear antigen (EBNA) in known EBV-associated cell lines was carried out. The ACIF procedure, in which the cell smears were thoroughly air-dried and fixed with carbon tetrachloride (for 15 min at room temperature), provided reproducible staining results with minimum variations. The fixed smears could be stored for at least 5 weeks at −8° C without loss of reactivity. On the basis of the above evaluation, a total of 59 human cell lines, which had unknown or uncertain association with EBV, were examined for the presence or absence of EBNA by the ACIF procedure. All of the cell lines tested could be judged positive or negative for EBNA. All of 21 lines growing as monolayer or in a mixed state of monolayer and suspension, were negative. Of 38 lines grown in suspension, 36 lymphoblastoid lines were positive but the remaining 2 lines (seemingly not lymphoblastoid) were negative.

Yorio Hinuma

Papers

Characteristics of cell lines derived from human leukocytes transformed by different strains of Epstein-Barr virus.

Identification of the target cells in human B lymphocytes for transformation by epstein-barr virus

Clonal Transformation of Human Leukocytes by Epstein-Barr Virus in Soft Agar

Simultaneous presence of EBNA-positive and colony-forming cells in peripheral blood of patients with infectious mononucleosis.

Evaluation of Epstein-Barr virus-associated nuclear antigen with various human cell lines.