Effect of trauma of implantation of metastatic tumor in bone in mice

We studied the influence of surgical trauma to the iliac bone on the implantation of I. V. injected tumor cells, which formed tumor in the surgical wounds of 27/84 mice (32%). None of these mice or nonsurgical mice developed tumor in the opposite or uninjured pelvic bone (P < 0.0001). When different numbers (10⁵, 5 × 10⁵, and 10 × 10⁵) of TA3Ha cells were injected I. V. immediately after surgery, the frequency of tumor formation showed an increase (respectively, 32%, 63%, 71%). As the interval between induction of trauma and tumor cell injection was increased from 0 to 15 days, the frequency of tumor formation declined from 32% to 0%. These results suggest that the healing wound is a privileged site for experimental metastasis, particularly in the early stages. It is likely that the proteins in the blood clotting cascade are involved in local tumor implantation. © 1994 Wiley-Liss, Inc.     

The influence of surgical trauma on experimental metastasis

Influence of surgical trauma on experimental metastasis in healing wounds is investigated using a transplantable murine mammary carcinoma cell line, TA3Ha. Intravenous injection of 10(5), 10(6), and 2 x 10(6) TA3Ha cells into syngeneic Strain A mice led to liver or kidney tumor development in none of the 96, ten, and ten mice tested, respectively. In contrast, injection of 10(5) cells into mice immediately after hepatic wedge resection performed using milliwatt carbon dioxide laser and electrocautery resulted in tumor formation at the site of trauma in 21/37 (57%) and 25/52 (48%) mice, (P less than 0.001) respectively. Similar results were obtained in mice subjected to partial nephrectomy using the laser (nine of 18) and electrocautery (eight of 13). These results clearly demonstrate that surgical trauma renders a nonprivileged organ susceptible to experimental metastasis formation, and that at least in this model both laser and electrocautery have similar effects. Tumor cell injection 1, 7, and 10 days posthepatic surgery resulted in 36%, 20%, and 0% tumor formation, respectively, indicating that the earlier events in wound healing support tumor implantation and/or growth better than those later on. Frequency of tumor formation at sites of trauma in the peritoneum induced by scalpel blade, laser, and electrocautery were 28%, 50% and 82%, respectively. Peritoneal tumors were seen in 33% of the nonsurgical mice. Skin incisions induced with the three above probes had little influence on experimental metastasis formation. Thus the influence of trauma on tumor formation is not uniform in every organ.     

The plasmin system in human adenocarcinomas and their metastases. A comparative immunofluorescence study

Components of the plasmin system were comparatively studied in lymph node metastases and corresponding primary tumors by immunofluorescence. Primary tumors, all adenocarcinomas, originated from large bowel (N = 12) or breast (N = 10). We used antisera against plasminogen (Pg), plasminogen activators (PA) such as urokinase (UK) and tissue type PA (t-PA), plasmin inhibitors such as alpha 2 anti-plasmin (alpha 2AP) and alpha 2 macroglobulin (alpha 2M), plasmin-alpha 2 anti-plasmin complex (PAPC). Positivity with anti-PAPC serum was considered as proving that plasmin was formed by Pg activation. The following results were obtained. Breast adenocarcinomas were more strongly stained than colorectal adenocarcinomas using antisera against Pg, PAPC and PA, while their reactivity was much weaker with antisera against both plasmin inhibitors. Lymph node metastases from colorectal adenocarcinomas were more strongly stained than primary tumors using antisera against PAPC and PA. Reactivity with anti-Pg was similar, while that with antisera against plasmin inhibitors was much weaker. Metastasis from breast adenocarcinomas, on the average, showed the same type of staining as primary tumors. However, there was a slight decrease in reactivity with anti-Pg and PAPC in metastases. Tumor cells invading lymphoid areas in metastatic lymph nodes were often strongly labeled using antisera against Pg and UK. Staining was less strong or less frequent using antisera against PAPC and t-PA. These results favor the role of plasmin in the degradation of basement membrane and connective tissue components, thus implicating it in the invasiveness of tumor cells, at least in most primary tumors and metastases.     

Relationship between circulating plasminogen activators and tumor development in mice

Plasminogen activators (PAs) present in the plasma of BALB/c mice and produced in vitro by murine tumor cell cultures (B77-3T3, SR-BALB, AA6) have been characterized using electrophoretic-zymographic techniques. BALB/c mouse plasma contains a main PA activity with an approximate molecular weight of 88,000 and pI 6.3, inhibited by anti-human tissue-type plasminogen activator (t-PA) serum, here defined as murine t-PA. On the contrary, all tumor cells tested release a PA activity with a molecular weight of 44,500 and pI 9.2 characteristic of urokinase-type activator (murine u-PA). The injection s.c. of the different tumorigenic cells into BALB/c mice leads to tumor development and to a rapid increase of t-PA from the first day following the injection. This early enhancement of t-PA activity is not detectable in mice given injections of lethally irradiated B77-3T3 cells. Moreover the development of the tumor in the animals is related to the appearance of increasing levels of u-PA in the blood. This activity is detectable in the plasma of treated mice almost 2 wk before detection of a tumor 1 mm in diameter. During tumor development, the molecular weight of the u-PA and t-PA forms present in the plasma does not change, while there is a decrease of the isoelectric point of the u-PA leading to the appearance of distinct PA activities with pI 7.6 and 8.9. Syngenic and allogenic lymphocytes, injected in BALB/c mice, do not induce any modification in the pattern of the plasma PA. The injection of highly metastatic cells, as opposed to nonmetastatic or low-metastatic cells, does not give rise to detectable levels of u-PA in the plasma of treated mice. These data suggest that the lack of plasma u-PA activity facilitates the formation of metastates, while the increase of this activity is important in tumor development and is independent of the metastatic potential of the injected cells.     

Fibrinolysis activity promotes tumor invasiveness of B16 melanoma cell lines through a reconstituted gel matrix.

We studied the role of the fibrinolytic function in the invasiveness of murine melanoma B16F1 and F10 cells using a reconstituted matrix on a filter in a modified Boyden chamber. The main species of plasminogen activators (PAs) synthesized in cell lysates and released into conditioned media by these cells was found to be tissue-type PA (t-PA). The invasiveness of these cell lines was enhanced by adding plasminogen to the gel matrix. This enhancing effect of plasminogen was markedly suppressed by adding anti-t-PA IgG and plasmin inhibitors into the gel matrix, but less affected by anti-urokinase-type PA (u-PA) IgG, offering more evidence to the hypothesis that the activation of the fibrinolytic system by PAs plays an important role in the invasiveness of murine melanoma B16 cell lines, and indicating that t-PA contributed more than u-PA to the invasive potential of these cells into the pericellular matrix.     

Factors influencing the growth and metastatic behavior of tumors

The factors influencing the growth and metastatic behavior of experimental animal tumors are examined. 10(4) TA3Ha cells were injected intraperitoneally, intravenously, subcutaneously into the flank, and subcutaneously into the tail tissues of syngeneic strain A mice. The tumor takes from these injections were 50/50 (100%), 1/10 (10%), 10/10 (100%), and 7/12 (60%), respectively. The frequency of lung metastasis from these sites was 0, 100, 50, and 100%, respectively. At the time of host death, the flank and tail tumors were, respectively, 2.2 +/- 0.5 cm (geometric mean diameter) and 1.2 +/- 0.3 cm in the TA3Ha and 0.9 +/- 0.1 cm and 0.4 +/- 0.1 cm in the L1210 systems. TA3Ha tumors metastasized regularly to the lymph nodes but the L1210 tumors seldom metastasized to the lymph nodes. Tail implants of TA3Ha tumors behaved similarly in the athymic nude mice and strain A mice. TA3Ha cells inoculated into the Millipore chambers and maintained in mice for greater than 150 days were viable and able to form tumors. The results demonstrate that the anatomic location of the tumor affects the growth and metastatic behavior of the tumors, and that the tumors of different histologic origin metastasize differently even when grown in corresponding locations.     

Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma

The biochemical events associated with tumor invasion involve localized degradation of the basement membrane by tumor-associated proteinases. In this study, we have characterized the proteinase secretion profiles of 5 ovarian epithelial carcinoma cell lines (DOV 13, OVCA 420, OVCA 429, OVCA 432, OVCA 433) as well as normal ovarian epithelial cells. Immunocapture assays demonstrated that all 5 carcinoma cell lines produce both secreted and surface-associated plasminogen activator. Urinary-type plasminogen activator (u-PA) production was one order of magnitude greater than production of tissue-type plasminogen activator (t-PA). Furthermore, t-PA secretion by normal ovarian epithelial cells was not detectable, whereas u-PA production was 17-to 38-fold lower than in ovarian carcinoma cells. Western-blotting analysis demonstrated that u-PA was secreted as the single chain form (scu-PA) when cells were cultured in serum-free medium. Incubation of plasminogen with ovarian carcinoma cell-conditioned medium resulted in direct activation of the Zymogen to plasmin. Furthermore, following incubation of cells with plasminogen, plasmin was eluted from the cell surface, indicating that ovarian carcinoma cells contain binding sites for plasminogen/plasmin which are accessible to surface-associated plasminogen activators. In addition to plasminogen activators, metalloproteinases were also produced by DOV 13, OVCA 429 and OVCA 433 cells. DOV 13 cells produce a 68-kDa metalloproteinase similar to matrix metalloproteinase 2 (MMP-2) whereas a 92-kDa enzyme similar to MMP-9 is secreted by OVCA 429 and 433. Together, ovarian carcinoma-associated plasminogen activators and metalloproteinases catalyze the hydrolysis of the major basement membrane protein components, type-IV collagen, type-IV gelatin, laminin and fibronectin. The enhanced proteolytic capability of ovarian carcinoma cells relative to normal ovarian epithelium suggests a biochemical mechanism by which invasion and spread of ovarian epithelial carcinoma may be mediated.     

Proteases during the growth of Ehrlich ascites tumor. I. The fibrinolysin system.

Component levels of the fibrinolysin system in the plasma and ascitic fluid of Swiss mice bearing Ehrlich ascites tumor cells were determined during a 15-day tumor growth time phase. During tumor growth, the concentration of plasminogen in the ascitic fluid decreased inversely to the total packed cell volume. Free plasmin was not present in the ascitic fluid nor was there any measurable plasminogen activator activity. Both antiplasmin activity and fibrinogen levels present in the fluid decreased during tumor growth. The nuclear and mitochondrial-microsomal subcellular fractions of the tumor cell exhibited plasminogen activator activity. No significant changes in the above parameters occurred in the plasma during the tumor growth period we studied.     

Rat mammary carcinoma cells secrete active collagenase and activate latent enzyme in the stroma via plasminogen activator

A spontaneous mammary adenocarcinoma (AC) from an inbred female rat was investigated with regard to secretion of neutral proteases. Cultures of neoplastic epithelial cells derived from the tumour secreted an enzyme that fulfilled the criteria for a specific collagenase. In contrast to cultures of non-neoplastic cells, tumour collagenase was present as an active enzyme, since treatment with trypsin or p-aminophenylmercuric acetate (APMA) did not increase activity. The neoplastic cells were also prolific producers of plasminogen activator (PA). Dexamethasone (Dex) (10(-6)M) markedly reduced the levels of both enzymes. Addition of tranexamic acid (TA), an inhibitor of plasmin and of plasminogen activation, did not affect collagenase activity, even at 10(-1)M TA, nor did latent collagenase accumulate. Latent collagenase was secreted in culture by normal fibroblasts from neonatal rat lungs. This latent enzyme was activated by the addition of tumour cell medium plus plasminogen, but this effect was inhibited by the addition of TA. These results demonstrate that the neoplastic cells themselves secrete collagenase as an active enzyme. PA is also secreted, is not involved with tumour collagenase, but is capable, in the presence of plasminogen, of activating latent collagenase produced by the non-neoplastic cells within the tumour or in the surrounding tissue. This tumour possesses potent collagenolytic ability in vitro which may be partly responsible for its rapid invasion in vivo.     

Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity

Three human tumor cell lines, Bowes' melanoma, HT1080 and Osmond cells, were characterized for their ability to invade the amniotic membrane and their production of plasminogen activator. Bowes' melanoma cells, which release large amounts of tissue plasminogen activator (tPA), were poorly invasive on the amniotic membrane. The addition of plasmin inhibitors, anti-tPA antibody or tissue inhibitor of metalloproteinase (TIMP) to the amnion assay enhanced invasiveness. The depletion of plasminogen from the growth medium also enhanced the degree of invasiveness. Similarly, HT1080 cells, which produce high levels of urokinase-type plasminogen activator (uPA), were poorly invasive under standard conditions but invasion was enhanced by plasmin inhibitors or anti-uPA antibodies. Conversely, Osmond cells, which produce low levels of uPA, were very invasive on the amniotic membrane. Invasion by these cells was blocked by the addition of plasmin inhibitors or anti-uPA antibodies to the amnion assay. These results suggest that invasion requires only a minimum level of PA activity and that, as PA production exceeds this optimal level, the degree of invasion decreases. We propose that high levels of plasmin, generated by the tPA or uPA secreted by the cells, may cause uncontrolled matrix degradation and interrupt the interaction of cells and matrix in the early stages of invasion. The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts and result in an enhancement of invasion.     

Partial nephrectomy in mice with milliwatt carbon dioxide laser and its influence on experimental metastasis

We have developed a surgical model to perform partial nephrectomy in mice using the milliwatt CO2 laser and have used this model for studying the influence of the sequel of surgery on experimental tumor metastasis. Strain A mice were subjected to partial nephrectomy using the milliwatt CO2 laser. The surgical procedure was time efficient, the blood loss was minimal, and the postoperative mortality was 6%. Immediately after surgery, the wound consisted of a superficial layer of charring and a deeper layer of thermal damage (coagulative necrosis). The wound healing was completed within 30 days and was accompanied by fibroblast infiltration and tubular regeneration but minimal inflammatory response. Seventy surgical mice were injected I.V. with TA3Ha murine mammary adenocarcinoma cells at different intervals (immediately to 30 days) after surgery. Among 38 mice inoculated with tumor cells immediately or up to 3 days after surgery, 18 (47%) showed histologically confirmed tumors at the site of surgical trauma. None of the 38 unoperated kidneys showed any evidence of tumor. This difference is statistically significant at a P value of less than 0.001. As the interval between surgery and tumor inoculation was increased to 7, 15, and 30 days, the frequency of tumor formation at the site of surgery decreased to 20% (2/10), 14% (2/14), and 0% (0/8), respectively. The results demonstrate that a) partial nephrectomy in mice is feasible with minimal mortality or apparent morbidity, b) the laser-induced surgical trauma favors implantation and growth of tumors, c) the frequency of tumor formation is related to the stage of wound healing, and d) the tumors are anatomically related to the healing wound but do not invade into the parenchymal tissue.     

The plasmin system in human colonic tumors: an immunofluorescence study

We studied the plasmin system with specific antisera to plasminogen, its 2 activators (urokinase-type and tissue-type) and the 2 plasmin inhibitors, alpha 2 anti-plasmin and alpha 2 macroglobulin on sections of 34 human colonic tumors by immunofluorescence. Anti-plasminogen serum showed a clear-cut reactivity at the surface of tumor cells, as it stained the contour of tumor glandular structures, foci, and isolated tumor cells. Intratumoral deposits and necrotic areas were stained as well, often strongly. Localization of plasminogen was quite different from that of fibrinogen, which was found only in peritumoral stroma, and never on tumor cells. Traces of both types of plasminogen activator were found, mainly on invasive tumor cells for urokinase type and on large tumor foci for tissue type. Images were weak and inconstant. Large amounts of both plasmin inhibitors were characterized in tumor stroma. Alpha-2 anti-plasmin was also found in intratumoral deposits and necrotic areas. It seems likely that plasminogen exudes from blood capillaries (since anti-plasminogen serum often stained the whole capillary wall), diffuses in the stroma and binds to tumor cells. Once formed, plasmin is likely to play a role in the invasion of surrounding tissues by tumor cells, in the dissociation of tumor cells from tumor glands and in the production of necrosis inside tumor areas.     

乳腺癌纤溶分子标志物t-PA、u-PA表达的临床研究

目的:检测乳腺癌患者血浆纤溶分子标志物含量、mRNA水平的变化以探索其与肿瘤浸润、播散的关系及临床意义。方法:采用逆转录、实时定量PCR技术检测30例乳腺癌组织中组织型纤溶酶原激活剂(tPA)、尿激酶型纤溶酶原激活剂(uPA)mRNA水平;用ELISA法同步检测患者血浆纤溶分子标志物含量,包括tPA、uPA、尿激酶型纤溶酶原激活剂受体(uPAR)、纤溶酶抗纤溶酶复合物(PAP)等。结果:乳腺癌患者血浆uPA、uPAR、PAP含量均较正常对照明显升高,其中,有周围淋巴结转移、远处脏器转移者uPA升高更为显著。肿瘤细胞uPAmRNA表达增高,在不同临床分期组中有显著差异,而tPAmRNA则减少。雌孕激素受体双阳性组tPAmRNA表达较其他组为高。结论:乳腺癌患者纤溶功能明显亢进,可能是其易播散、浸润的主要原因之一。逆转录实时定量PCR技术,使tPAmRNA、uPAmRNA有望成为临床上乳腺癌病情监测、预后判断及治疗选择的辅助指标。     

Measurement of plasminogen activator activity from human fibrosarcoma cells by a new microassay

Elevated levels of plasminogen activator (PA) activity have been correlated with neoplasia and may have an important role in tumor-cell invasion and metastasis. We have developed a new caseinolytic assay that uses an immunochemical approach to measure the activity of PA elaborated by malignant tumor cells. The highly sensitive assay consists in incubating a source of PA (viable tumor cells, cell extracts, or conditioned medium) with purified plasminogen in microtiter plates precoated with a suitable protein substrate such as casein. Clearance of the immobilized protein substrate by PA-generated plasmin is then measured by a technique based on the enzyme-linked immunosorbent assay. In experiments using urokinase as a source of PA, the assay displayed near linearity over several log units of urokinase activity and could detect as little as 10(-2) Ploug units of PA activity. Besides successfully measuring PA activity produced by the human HT 1080 fibrosarcoma cell line, the assay permitted detection of significant plasminogen-independent proteolytic activity generated by intact tumor cells cultured in direct contact with immobilized protein substrates.     

Induction of morphological transformation, anchorage-independent growth and plasminogen activators in non-tumorigenic human osteosarcoma cells by lead chromate

The possible carcinogenicity of insoluble chromium (VI) compound, PbCrO4, in human cells has been tested using a nontumorigenic human osteosarcoma cell line (HOS, TE 85). Electron microscopic studies show that PbCrO4 is phagocytosed by HOS cells and accumulates within the vacuoles in the cytoplasm. A number of cell lines have been isolated following multiple treatment of HOS cells with PbCrO4. These cell lines are morphologically different from HOS cells, form anchorage-independent colonies in soft agar and form quickly regressing small tumor nodules in athymic nude mice. The cellular and secreted plasminogen activator (PA) levels of 5 cell lines isolated after PbCrO4 treatment are increased up to 8 fold and up to 10 fold respectively as compared to untreated HOS controls. SDS-PAGE analysis in the presence of copolymerized substrates is consistent with increase in 55 kDa urokinase-type PA (u-PA) and 68 kDa tissue-type PA (t-PA). These results show that PbCrO4 treatment leads to stable phenotypic changes indicative of the transformation of HOS cells.     

基质降解酶u-PA,PAI-1与人肺癌细胞浸润转移的相关关系的研究

观察体外细胞株ＰＧ、ＰＡａ细胞的ｕ－ＰＡ，ＰＡＩ－１活性和抗原表达，结果发现ＰＧ、ＰＡａ细胞均能够产生ＰＡ及ＰＡＩ，免疫细胞化学法定位ＰＡ主要为ｕ－ＰＡ，ＰＡＩ主要为ＰＡＩ－１。在ＰＧ为ｕ－ＰＡ高表达，而ＰＡａ为低ｕ－ＰＡ表达，ＰＡＩ－１在ＰＧ、ＰＡａ细胞中均高表达。对ＰＧ瘤细胞在ＢＡＬＢ／ＣＡ裸鼠皮下种植性瘤组织的免疫组化结果显示，ｕ－ＰＡ阳性～强阳性表达，ＰＡＩ－１为阴性表达。说明ｕ－ＰＡ是ＰＧ、ＰＡａ高低不同的转移能力的重要机制之一。     

Plasminogen activators play an essential role in extracellular-matrix invasion by lymphoblastic T cells

Involvement of extravascular sites, in particular infiltration of the central nervous system, is a frequent complication of T-lymphoblastic leukemia and contributes to leukemia-associated morbidity. In this report, we studied the contribution of plasminogen activators to the invasive properties of 7 human T-cell lines in a model of transmigration through an extracellular matrix. The T-cell lines were found to express either urokinase (u-PA) and high levels of u-PA receptor or tissue-type plasminogen activator (t-PA) and low levels of u-PA receptor. The rate of transmigration was consistently higher for u-PA-expressing cells than for t-PA-expressing cells. PA-inhibitor type I (PAI-1) was detected in the conditioned medium of one cell line and PAI-2 was detected in cell extracts from 6 lines. The transmigration of 6 out of 7 cell lines was inhibited by trasylol, an inhibitor of plasmin, by an excess of exogenous PAI-1 or PAI-2, and by antibodies to the particular PA type expressed by the cells. Partial inhibition of transmigration by the amino-terminal fragment of u-PA implies that the u-PA receptor contributes to transmigration. Thus, the transmigration of T-leukemia cells through a barrier of extracellular matrix requires PA-dependent proteolysis, which can be provided either by u-PA or t-PA. Specific inhibition of the PA system could provide a means to inhibit tissue invasion by T lymphoblastic cells. Int. J. Cancer, 70:461–466, 1997. © 1997 Wiley-Liss, Inc.     

Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation. Int. J. Cancer 73:740–748, 1997. © 1997 Wiley-Liss, Inc.     

Expression of urokinase-type plasminogen-activator (upa) and its receptor (upar) in human ovarian-cancer cells and in-vitro invasion capacity

Expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), their inhibitor PAI-1 and the uPA-receptor (uPAR) was characterized in six human tumor cell lines (OV-MZ-6, -10, -13, -15, -19 and OVCAR-3) established from patients with cystadenocarcinoma of the ovary. The invasive potential of the ovarian cancer cell lines determined in an in vitro invasion assay did neither correlate with the antigen level of uPA, t-PA, PAI-1 or uPAR nor with the cell surface uPA activity, however, did correlate with the cell surface-bound plasmin activity. The in vitro invasiveness of three cancer cell lines selected displaying a different pattern of uPA and uPAR expression was significantly inhibited by a recombinant soluble truncated form of the uPAR functioning as a scavenger for uPA. Our results suggest that the interference of the uPA/uPAR interaction leads to a reduced in vitro invasiveness of human ovarian cancer cells independent of the level of uPA and uPAR expression.     

Plasminogen activators in breast-cancer cells

This review summarizes recent experimental and clinical studies on plasminogen activator (PA) expression in breast cancer cells. The two PAs, urokinase-type (u-PA) and tissue-type (t-PA), have quite different biological significance in breast cancer cells. Sufficient evidence indicates that t-PA production is regulated by estrogen via an estrogen receptor system and suggests the potential usefulness of this enzyme as a marker for estrogen action in breast cancer cells. On the other hand, u-PA has been implicated in cancer cell invasion and metastasis formation because this enzyme can degrade components of the extracellular matrices and basement membranes either directly through its enzyme activity or indirectly by activation of other proteinases. High levels of u-PA correlates with both shortened disease-free survival and overall survival in patients with breast cancer indicating that u-PA is a new prognostic marker in human breast cancer. In contrast to u-PA, t-PA is associated with a good prognosis. However, t-PA is involved in bone-only metastasis formation by its effects through the vertebral venous plexus.