High Levels Of Plasminogen Activator Research Articles

The new DR‐70 immunoassay detects cancer of the gastrointestinal tract: a validation study

Malignant cells characteristically possess high levels of plasminogen activator, which induce local fibrinolysis. The DR-70 immunoassay is a newly developed test, which quantifies fibrin degradation products in serum by a proprietary antibody. To evaluate the DR-70 immunoassay as a detection assay for the presence of gastrointestinal cancers. We prospectively collected blood sera of 85 patients with histologically proven tumour and 100 healthy blood donors. Ten microlitres of the sera was used for the DR-70 immunoassay. Nineteen patients had a hepatocellular and 10 cholangiocellular carcinoma, 13 cancer of the pancreas, 30 colorectal cancer, 10 stomach cancer and three cancer of the oesophagus. Receiver-operator curve analysis revealed <0.7 microg/mL as the best cut-off value to distinguish between patients with cancer and healthy controls. Using this cut-off value, the DR-70 immunoassay showed a good clinical performance with a sensitivity of 91% and a specificity of 93%. Patients with advanced tumour spread showed significantly higher DR-70 values than those with early-stage tumours (P < 0.0003). The DR-70 immunoassay reliably differs between cancer patients and healthy controls. Therefore, it promises to become a useful test for the detection of cancer in clinical practice.

Bacterial O6-methylguanine-DNA methyltransferase reduces N-methyl-N'-nitro-N-nitrosoguanidine induction of plasminogen activator in Mer- human glioblastoma A1235 cell line.

The alkylation repair deficient (Mer- phenotype) cells produce high levels of proteolytic enzyme plasminogen activator (PA) after treatment with alkylation agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Both, in Escherichia coli and in mammalian cells O6-methylguanine (O6-MeG) is repaired by analogues O6-methylguanine-DNA methyltransferase (MGMT). In E. coli MGMT is product of ada gene. To investigate the effect of bacterial MGMT expression on the induction of PA activity in human cells, we have transfected ada-alkB operon into Mer- human A1235 cells that are known to produce high levels of PA after MNNG treatment. We have shown here that A4 and A8 transformants that harbour ada gene become resistant to killing by MNNG. In addition, MNNG produced induction of extracellular PA activity was much less pronounced in A4 and A8 transformants (induction ratio 3.42 and 3.74, respectively) than in control A1235 and Aneo-1 cells (induction ratio 11.04 and 9.11, respectively). However, changes of intracellular PA activity were not significant. It appears, therefore, that induction of extracellular PA activity is inversely related to the cell capacity to repair the DNA lesions induced by alkylation agents.

Growth and Dissemination of Lewis Lung Carcinoma in Plasminogen-Deficient Mice

Plasminogen activation has been proposed to play a critical role in cancer invasion and metastasis. The effects of complete ablation of plasminogen activation in cancer was studied by inoculation of a metastatic Lewis lung carcinoma expressing high levels of plasminogen activator into plasminogen-deficient (Plg−/−) mice and matched control mice. Primary tumors developed in all mice with no difference in the rate of appearance between Plg−/− and control mice. However, the primary tumors in Plg−/− mice were smaller and less hemorrhagic and displayed reduced skin ulceration. In addition, dissemination of the tumor to regional lymph nodes was delayed in Plg−/− mice. Surprisingly, no quantitative differences were observed in lung metastasis between Plg−/− and control mice. In addition, Plg deficiency was compatible with metastasis of the primary tumor to a variety of other organs. Nevertheless, Plg−/− mice displayed a moderately increased survival after primary tumor resection. These findings suggest that plasmin-mediated proteolysis contributes to the morbidity and mortality of Lewis lung carcinoma in mice, but sufficient proteolytic activity is generated in Plg−/− mice for efficient tumor development and metastasis.

Growth and Dissemination of Lewis Lung Carcinoma in Plasminogen-Deficient Mice

AbstractPlasminogen activation has been proposed to play a critical role in cancer invasion and metastasis. The effects of complete ablation of plasminogen activation in cancer was studied by inoculation of a metastatic Lewis lung carcinoma expressing high levels of plasminogen activator into plasminogen-deficient (Plg−/−) mice and matched control mice. Primary tumors developed in all mice with no difference in the rate of appearance between Plg−/− and control mice. However, the primary tumors in Plg−/− mice were smaller and less hemorrhagic and displayed reduced skin ulceration. In addition, dissemination of the tumor to regional lymph nodes was delayed in Plg−/− mice. Surprisingly, no quantitative differences were observed in lung metastasis between Plg−/− and control mice. In addition, Plg deficiency was compatible with metastasis of the primary tumor to a variety of other organs. Nevertheless, Plg−/− mice displayed a moderately increased survival after primary tumor resection. These findings suggest that plasmin-mediated proteolysis contributes to the morbidity and mortality of Lewis lung carcinoma in mice, but sufficient proteolytic activity is generated in Plg−/− mice for efficient tumor development and metastasis.

In vitro production of plasminogen activator by human granulosa cells.

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.

Serum factors responsible for unusual induction of plasminogen activator activity in tuberous sclerosis

Peripheral blood lymphocytes derived from tuberous sclerosis (TS) patients showed unusually high levels of plasminogen activator (PA) activity after treatment with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Serum obtained from peripheral blood of TS patients also enhanced the PA activity level when normal contro lymphocytes were incubated with the serum prior to MNNG treatment. Factors exhibiting the enhancing activity were eluted with a solution of about 0.70 M KCl on dye-ligand chromatography, which were inhibited on incubation with an anti-human interferon (HuIFN)-β antibody, but not with anti-HuIFN-α or anti-HuIFN-γ antibodies. Unlike in the case of HuIFN-β, the eluted samples did not possess antiviral or anticellular activity. Thus, it seems likely that serum from TS patients contains factors which are responsible for the unusual PA induction and which have a similar epitope to HuIFN-β.

Bony ankylosis of the temporomandibular joint: Case report of a child treated with delrin condylar implants

primary fibrinolysis. This theory gained further acceptance after the demonstration of high levels of plasminogen activator in prostatic tissue.6 It is believed that this excess plasminogen activator is produced as a result of surgical trauma to prostatic tissue, resulting in a hypocoagulability of the blood.7 More recent clinical studies have suggested that thromboplastins released from prostatic tumor cells initiate a disseminated intravascular coagulation (DIC) syndrome, with fibrinolysis occurring secondarily. It is estimated that 25% of cases of chronic DIC have an underlying carcinoma of the prostate.8 DIC was ruled out in this case. Systemic use of l -aminocaproic acid, a drug that can effectively prevent clot dissolution by inhibiting the activation of plasminogen by plasminogen activators, was considered in the treatment of the patient. However, it was not used because of the consulting physician’s fears that it could contribute to systemic thrombosis and because the patient’s condition appeared to be improving. However, the local or systemic use of an antifibrinolytic agent for such cases warrants further investigation.

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Identification of t-PA as the Major Active Plasminogen Activator in Human Milk

Human colostrum and milk contain high levels of plasminogen activator when compared with blood. These levels are inversely related to the duration of lactation and the activity appears to be due to tissue type plasminogen activator present in multiple (65-190 Kd) molecular weight forms.

Chromosomal locations of human tissue plasminogen activator and urokinase genes.

A panel of human-mouse somatic cell hybrids and specific complementary DNA probes were used to map the human tissue plasminogen activator and urokinase genes to human chromosomes 8 and 10, respectively. This result is in contrast to a previous assignment of a plasminogen activator gene to chromosome 6. As neoplastic cells produce high levels of plasminogen activator, it is of interest that aberrations of chromosome 8 have been linked to various leukemias and lymphomas and that two human oncogenes, c-mos and c-myc, have also been mapped to chromosome 8.

The transformed phenotype in culture and tumorigenicity of fischer rat fibroblast cells (FR3T3) transformed with bovine papilloma virus type 1

Unlike cell lines transformed with Polyomaviruses, transformants derived by focus formation or colony formation in agarose medium after transfer into rat fibroblast cells (FR3T3 line) of Bovine Papilloma Type 1 (BPV1) DNA were consistently observed to grow poorly in suspension and to remain highly serum dependent for growth in culture. These cells did not produce detectable amounts of plasminogen activator, and kept the flat morphology and organized cytoskeleton characteristic of the normal fibroblast. However, they induced in syngeneic animals the development of tumors with a greater invasive potential than tumors induced by Polyomaviruses. By contrast with the original transformants, cells recovered from the tumors grew efficiently in suspension and produced high levels of plasminogen activator. They still had, however, extended cytoskeletal structures and remained completely dependent on high serum concentrations for growth in culture. The stepwise transformation process induced by BPV1 thus appears strikingly different from that previously observed with polyoma and SV40 viruses. The observed changes in transformation phenotype between transformed line and tumor cells do not correlate with any important modification of the number of autonomous copies of the viral genome, nor with any rearrangement of viral sequences detectable at the level of the blot analysis.

ChemInform Abstract: PLASMIN‐ACTIVATED PRODRUGS FOR CANCER CHEMOTHERAPY. 1. SYNTHESIS AND BIOLOGICAL ACTIVITY OF PEPTIDYLACIVICIN AND PEPTIDYLPHENYLENEDIAMINE MUSTARD

AbstractDurch Umsetzung der Verbindungen (I) und (II) in Gegenwart von Dicyclohexylcarbodiimid (DCC) und I‐Hydroxybenzotriazol (HOBT) entsteht das Peptid (III), das über (IV) in die "Prodrugs" (V) und (VI) übergeführt werden kann.

ChemInform Abstract: PLASMIN‐ACTIVATED PRODRUGS FOR CANCER CHEMOTHERAPY. 2. SYNTHESIS AND BIOLOGICAL ACTIVITY OF PEPTIDYL DERIVATIVES OF DOXORUBICIN

AbstractDie Verbindung (I) und das 9‐Fluorenylmethoxycarbonyl‐Derivat (II) reagieren zum Peptid (H I), das in das Doxorubicinpeptid (V) übergeführt wird.

Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard

Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, we synthesized the peptidyl prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection with trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. Furthermore, the prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. These results suggest that plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.

Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin

We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.

Dextran-induced lowering of plasminogen proactivator and functionally active high molecular weight kininogen in the rat.

Incubation of plasminogen-free rat citrated plasma with acetone (23% v/v) yielded enzyme preparations with high levels of plasminogen activator (PGA) and kininogenase (kallikrein), but with a low concentration of high molecular weight kininogen (HMWK) active as cofactor for kaolin-induced activation of factor XII. When benzamidine (4.0 mM) was present during acetone activation, a high yield of functionally active HMWK was obtained. Gel chromatography separated PGA into one high molecular weight fraction (HMW-PGA) without kininogenase and BAEe esterase activity, and one fraction (LMW-PGA) eluting together with plasma kallikrein. Injection of dextran (100 mg/kg intravenously) reduced the amount of LMW-PGA to 40%, without altering the concentration of HMW-PGA, and with only a small reduction of the kininogenase activity.

Expression of transformation-associated traits in the myogenic cell lines L6 and L8

The presence of cellular alterations, usually associated with transformation, has been studied in two permanent myogenic cell lines, L6 and L8, that retain the ability to differentiate in vitro. We present evidence that, beside being immortal, both cell lines are anchorage-independent for proliferation, a feature not found in primary muscle cells. L6 secretes constitutively high levels of plasminogen activator. L8 is able to undergo multinucleation in the presence of cytochalasin B (cytB) and is tumorigenic in vivo. Single anchorage-independent clones were shown to possess differentiative potentials similar to those of the parental line. Moreover, cell fusion could be directly observed in L8 while still growing as colonies in soft agar. We discuss our data with respect to ( i) the reported differences in the regulation of differentiation between primary myogenic cells and continuous cell lines; ( ii) the relationship between transformation and differentiation in muscle cells.

Clonal variation in cultured neuroblastoma cells. I. Isolation and characterization of variants.

Two spontaneously arising variant clones were selected from the N18 neuroblastoma cell line solely on the basis of their flattened morphology and tight adherence to the culture flask. Two other clones having the round loosely adherent morphology typical of the parent line were also selected, and flat variants were shown to arise in them upon prolonged cultivation. The flat variant clones have slower growth rates in culture, lower cloning efficiencies in suspension, and reduced acetylcholinesterase inducibility when compared with either the parent N18 line or the round cell clones. Cells of both morphologic types have high levels of plasminogen activator and are tumorigenic, although the variants have a slower growth rate in vivo, consistent with their slower growth rate in culture. SDS-polyacrylamide gel electrophoresis of total protein from the two cell types shows that the flat variants have increased amounts of a 200,000 molecular weight polypeptide that has tentatively been identified as the heavy chain of myosin. Round morphological revertants from one of the flat variant clones exhibited growth characteristics typical of the parent N18 line, but their content of myosin heavy chain, although reduced, was not so low as that in the round cell clones originally isolated. The possibility of a causal relationship between flat morphology, reduced suspension cloning efficiency, and increased content of myosin heavy chain is discussed.

Serum-mediated suppression of cell-associated plasminogen activator activity in cultured endothelial cells

Cultured bovine aortic endothelial cells accumulate high levels of plasminogen activator (PA) activity at confluency. This cell-associated PA (PA c) activity remains high when the cells are maintained without refeeding. Addition of fresh growth medium to these cultures results in a 70–80% decrease in this activity. The decrease is apparent within 30 min, is complete by 2 hr and is initiated by the fetal calf serum (FCS) in the medium. The decrease is transient, with PA c returning to control levels within 4 days, and occurs each time these confluent cultures are fed; it is not associated with the initiation of DNA synthesis. The magnitude of the decrease in PA c activity depends on the concentration of the added serum, with 0.1% FCS causing a 50% decrease. Maximum suppression is observed with 1% FCS. The decrease in PA c activity is not due to serum-mediated stimulation of secretion since PA secretion is also suppressed by fresh serum. Neither changes in the activity of cellular inhibitors nor the presence of contaminating serum inhibitors appears to account for the observed loss of activity. The suppressor activity of serum is nondialyzable but heat-labile. Treatment of serum with diisopropylfluorophosphate (DFP) or hirudin does not reduce its suppressor activity, suggesting that neither thrombin nor other serine proteases are involved. This PA-suppressor activity is not a unique property of FCS since sera from other species, as well as bovine platelet-poor plasma (PPP), are effective in reducing PA c activity. These observations indicate that the fibrinolytic activity of cultured endothelial cells is modified by routine culture manipulations and suggest that production of PA by the endothelium itself may be subject to negative regulation by suppressor molecules in the blood.