Long-term correction of mouse dystrophic degeneration by adenovirus-mediated transfer of a minidystrophin gene.

Duchene muscular dystrophy (DMD) is a fatal progressive X-linked muscle disorder, caused by mutations in the dystrophin gene. We have investigated adenovirus-mediated transfer of a dystrophin minigene in a mutant mouse lacking dystrophin, the mdx mouse. We report here that six months after a single intramuscular injection of a recombinant adenovirus containing a human dystrophin minigene, a large number of dystrophin-positive fibres are still detected in the injected muscles. Moreover, although the minigene encodes a truncated protein, its expression is able to protect the fibres efficiently against the degeneration process that affects the dystrophin-deficient mdx myofibres.

Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

[Hemophagocytic syndrome and toxoplasmic primo-infection]. / Syndrome d'activation macrophagique et primo-infection toxoplasmique.

Hemophagocytic syndrome (HPS) is a clinical entity that combines the clinical, biological and histological symptoms. The physiopathological mechanism involves the interaction between T lymphocytes/NK cells and macrophages, at the origin of an uncontrolled activation of the macrophages. The consequence is a hemophagocytosis extending to numerous organs, preferentially bone marrow. Clinical symptoms include cytopenia, fever unresponsive to antibiotics and multiple organ dysfunctions. Infections, lymphoproliferative disorders, cancers, systemic diseases are the most prevalent triggers or etiologies of HPS. Because of its high risk of mortality, HPS constitutes a diagnostic and therapeutic urgency. The search for an aetiology, in particular by serological testing, is essential because it conditions the treatment and thus the evolution of the disease. We report here the case of a 12 years-old boy presenting a HPS secondary to a toxoplasmic primo-infection. The objective of this work is to present the step of the biological diagnosis of HPS. Moreover, this observation allows the study of a very rare clinical presentation of toxoplasmic primo-infection, in an immunocompetant patient.

ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis.

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

Widespread long-term gene transfer to mouse skeletal muscles and heart.

Successful treatment of muscular disorders awaits an adapted gene delivery protocol. The clinically applicable technique used for hematopoietic cells which is centered around implantation of retrovirally modified cells may not prove sufficient for a reversal of phenotype when muscle diseases are concerned. We report here efficient, long-term in vivo gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant adenovirus. This simple, direct procedure raises the possibility that muscular degenerative diseases might one day be treatable by gene therapy.

Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury.

The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal-truncated EGF-R under the control of the kidney-specific type 1 gamma-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.

Reduction of renal mass is lethal in mice lacking vimentin. Role of endothelin-nitric oxide imbalance.

Modulation of vascular tone by chemical and mechanical stimuli is a crucial adaptive phenomenon which involves cytoskeleton elements. Disruption, by homologous recombination, of the gene encoding vimentin, a class III intermediate filament protein mainly expressed in vascular cells, was reported to result in apparently normal phenotype under physiological conditions. In this study, we evaluated whether the lack of vimentin affects vascular adaptation to pathological situations, such as reduction of renal mass, a pathological condition which usually results in immediate and sustained vasodilation of the renal vascular bed. Ablation of 3/4 of renal mass was constantly lethal within 72 h in mice lacking vimentin (Vim-/-), whereas no lethality was observed in wild-type littermates. Death in Vim-/- mice resulted from end-stage renal failure. Kidneys from Vim-/- mice synthesized more endothelin, but less nitric oxide (NO), than kidneys from normal animals. In vitro, renal resistance arteries from Vim-/- mice were selectively more sensitive to endothelin, less responsive to NO-dependent vasodilators, and exhibited an impaired flow (shear stress)- induced vasodilation, which is NO dependent, as compared with those from normal littermates. Finally, in vivo administration of bosentan, an endothelin receptor antagonist, totally prevented lethality in Vim-/- mice. These results suggest that vimentin plays a key role in the modulation of vascular tone, possibly via the tuning of endothelin-nitric oxide balance.

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin.

The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

Hsp104 interacts with Hsp90 cochaperones in respiring yeast.

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Lipid metabolism and enzyme activities in hormone-dependent and hormone-independent mammary adenocarcinoma in GR mice.

Lipid metabolism in hormone-dependent (HD) GR mouse mammary tumors was compared to that in hormone-independent (HI) tumors and normal mammary tissues. HD tumors, like normal mammary tissue but unlike HI tumors, synthesized medium-chain-length fatty acids (MCFA). However, when treated with hormones (estrone and progesterone), the HI tumors were induced to produce MCFA. The activity of thioesterase II correlated positively with the synthesis of MCFA and was influenced by the hormones administered. The activities of NADP+-linked malate dehydrogenase, citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase, although lower in tumors than in normal glands, were not different in HD as compared to HI tumors. Whereas the predominating lipids synthesized in normal glands were triglycerides, phospholipids comprised about half of the lipid synthesized in the tumors, with no difference between HD and HI tumors. The conversion of D-[U-14C]glucose to 14CO2 was higher in HD tumors than in HI tumors but increased in HI tumors treated with hormones in vivo. By a comparison of the 14CO2 produced from D-[1-14C]glucose and from D-[6-14C]glucose in the presence and absence of an electron acceptor (methylene blue), it was demonstrated that regeneration of NADP+ from NADPH was a rate-limiting step for the pentose phosphate pathway in the tumors. Hence, while differences in the lipid metabolism can be demonstrated between HD and HI GR mouse mammary tumors, some of the changes are due to the hormone treatment rather than to a specific alteration in the tumor itself.

Effect of estrogen and antiestrogen on the human breast cancer cell line MCF-7 adapted to growth at low serum concentration.

The human breast cancer cell line MCF-7 has been adapted to long-term growth at 0.5 and 0.05% fetal bovine serum. Free cytoplasmic and filled nuclear estrogen receptors were found in cells grown at 5, 0.5, and 0.05% fetal bovine serum. Cells grown in medium with 0.05% dextran-charcoal-treated serum contained cytoplasmic receptors but no filled nuclear receptors, indicating that this medium did not contain biologically active concentrations of estrogen. Addition of estradiol to the medium translocated the cytoplasmic receptor to the nucleus and stimulated progesterone receptor synthesis but did not increase the growth rate. The antiestrogen tamoxifen (TAM) inhibited cell growth at all serum concentrations investigated, at least in part by a reduction of the growth fraction. The sensitivity to TAM was highest at low serum concentrations. The effect of TAM could be reversed by estradiol at TAM concentrations of 10(-6) M or lower. It is concluded that estradiol does not have a direct growth-stimulatory effect on our MCF-7 cells in monolayer cultures although the cells contain functional estrogen receptors and growth of the cells in athymic mice requires estrogens. TAM has an estrogen-competitive, inhibitory effect on the growth of MCF-7 cells at concentrations lower than 10(-6) M. At higher concentrations, the growth inhibition is unspecific and noncompetitive by estradiol.

The effect of estrone-progesterone treatment on cell proliferation kinetics of hormone-dependent GR mouse mammary tumors.

Hormone-dependent mammary tumors were induced in virgin GR mice by treatment with estrone and progesterone. Discontinuation of hormonal treatment was followed by regression of the tumor. This response to hormone treatment was also observed in the first transplant generation in inbred syngeneic hosts, but after several transplantations the tumor growth became hormone independent. The hormone dependence of the primary tumors and tumors after a single transplantation was demonstrated by growth curves. Furthermore, the cell proliferation kinetics has been investigated in a growing as well as in a regressing hormone-dependent tumor after a single transplantation from the same primary tumor. The experimental data consist of growth curves, percentage-labeled mitoses curves, and labeling indices. Since these data do not contain information concerning the localization of the cell loss in the cell cycle, they were analyzed by a computer method based on three mathematical models differing in respect to the mode of cell loss. All three models gave approximately the same estimates of the cell kinetic parameters in the growing as well as the regressing tumor. The results for the growing, hormone-dependent tumor showed a growth fraction of 62%, a cell production rate of 3.4%/hr, and a cell loss rate of 2.3%/hr. Regression of the tumor after hormonal deprivation was accompanied by a decrease in growth fraction to 18% and a decrease in the cell production rate to 0.9%/hr, while the cell loss rate was unchanged at 2.8%/hr. Furthermore, the discontinuation of hormonal treatment introduced an increase in the mean transit time of the cell cycle, particularly in the mean transit time of the G1 phase. The results might indicate that estrone and progesterone treatment stimulated growth of hormone-dependent GR mouse mammary tumors mainly by an increase of growth fraction and cell production rate.

Altered expression of estrogen-regulated genes in a tamoxifen-resistant and ICI 164,384 and ICI 182,780 sensitive human breast cancer cell line, MCF-7/TAMR-1.

A stable, tamoxifen-resistant subline, MCF-7/TAMR-1, of the human breast cancer cell line MCF-7 has been established in tissue culture after long-term treatment with 10(-6) M tamoxifen. The MCF-7/TAMR-1 cell line grows equally well in the presence and absence of tamoxifen, whereas the steroidal antiestrogens ICI 164,384 and ICI 182,780 exert profound inhibitory activity on cell proliferation, although higher concentrations are required to inhibit these cells compared to the parent cells. The MCF-7/TAMR-1 cells grown in tissue culture deviate from parent characteristics by the complete lack of expression of progesterone receptors even when grown with estradiol, by an altered tamoxifen regulation of M(r) 52,000 cathepsin D synthesis and secretion, and by lack of tamoxifen stimulation of an estradiol down-regulated M(r) 42,000 protein with presumed growth inhibitory function. MCF-7/TAMR-1 cells are estrogen receptor positive. The estrogen receptors have wild-type characteristics with respect to (a) binding of estradiol, tamoxifen, and ICI 164,384; (b) estrogen and antiestrogen regulation of the estradiol-regulated proteins pS2, M(r) 61,000 alpha 1-antitrypsin-like protein, M(r) 66,000 alpha 1-antichymotrypsin-like protein, and corresponding mRNAs; and (c) estrogen and antiestrogen regulation of a transiently transfected estrogen responsive reporter gene. We suggest that the lack of tamoxifen up-regulation of the M(r) 42,000 protein synthesis in MCF-7/TAMR-1 cells may at least partly explain the resistance to tamoxifen treatment. The sensitivity to the growth inhibitory activity of ICI 164,384 and ICI 182,780 may be ascribed to the maintenance of the pure antagonistic effect of these steroidal antiestrogens on MCF-7/TAMR-1 cells. Our results indicate that treatment with pure antiestrogens may be effective when patients become refractory to tamoxifen therapy.

The estrogen receptor variant lacking exon 5 has dominant negative activity in the human breast epithelial cell line HMT-3522S1.

The estrogen receptor variant lacking exon 5 (ERdeltaE5) encodes a truncated protein that lacks the hormone-binding domain and has been suggested to be responsible for the estrogen-independent growth of human breast tumors and resistance to antiestrogen therapy. The biological function of the ERdeltaE5 in human breast epithelial cells has been studied by transient transfection of HMT-3522S1 cells with wild-type (wt) estrogen receptor (ER) and ERdeltaE5. A 10-fold higher expression of ERdeltaE5 mRNA compared to that of wt ER mRNA was found. However, the expression of ERdeltaE5 protein was significantly lower than the expression of wt ER protein. The ERdeltaE5 was unable to activate the transcription of an estrogen-responsive reporter gene in the absence as well as in the presence of estrogen. The ERdeltaE5 disclosed a dominant negative activity when expressed together with wt ER. These data indicate that the biological significance of ERdeltaE5 in human breast cancer is dubious.

Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal.

We have reported previously on the first spontaneously immortalized, nonmalignant human breast epithelial cell line, HMT-3522, which is entirely dependent on exogenous epidermal growth factor (EGF). In passage 118, cells were adapted to grow in medium without EGF and a new growth-transformed subline, HMT-3522/gt-1, was generated and propagated at high growth rate without exogenous EGF (Madsen et al., Cancer Res., 52: 1210-1217, 1992). Here we have used this subline and the continuum of the parent line, HMT-3522/wt, to pose the question whether a relevant change in a physiological parameter of the microenvironment will induce malignant transformation. The two cell lines were cultured under identical conditions with the only exception that EGF was omitted in the medium for gt-1. Initially, wt and gt-1 were identical in terms of karyotype as well as morphology, growth rate, and protein expression as revealed by two-dimensional gel electrophoresis. A highly dramatic shift to phenotype was observed in passage 238 when the gt-1 line became tumorigenic in nude mice. After two mouse-culture passages, the resulting malignantly transformed cell line (HMT-3522/mt-1) was refractory to the growth-modulating effect of EGF and presented an extra copy of a chromosome marker, 7q-, as the only cytogenetic difference from the gt-1. Our results suggest that microenvironmental cues are powerful factors in the induction of malignancy. A major role of EGF receptor in the malignant transformation is emphasized by loss of EGF sensitivity and acquisition of an extra chromosome 7p harboring the EGF receptor gene. We hypothesize that during premalignant hyperplasia, a population of EGF/transforming growth factor alpha autonomous epithelial cells in situ may develop as a consequence of local transforming growth factor alpha deprivation, a condition reflected in the culture model as autonomy after EGF withdrawal.

Altered gene expression of c-myc, epidermal growth factor receptor, transforming growth factor-alpha, and c-erb-B2 in an immortalized human breast epithelial cell line, HMT-3522, is associated with decreased growth factor requirements.

Activation of protooncogenes and constitutive secretion of autocrine growth factors are thought to be involved in the uncontrolled growth of cancer cells. We have attempted to elucidate the role of oncogenes and growth factors in the premalignant progression of human breast epithelial cells by using an immortalized, nontumorigenic, near-diploid human mammary epithelial cell line, HMT-3522, derived from a fibrocystic lesion and established in our laboratory. During propagation in tissue culture, the growth factor requirements of the HMT-3522 cells decreased simultaneously with an amplification and overexpression of the c-myc protooncogene. Other protooncogenes related to human breast cancer were unaltered with regard to gene copy number and expression. In passage 118, in which the most important growth factor still was epidermal growth factor (EGF), we were able to isolate an EGF-independent subline (S2). The EGF independence of S2 was accompanied by an overexpression of the mRNAs for epidermal growth factor receptor (EGF-R), transforming growth factor-alpha, and c-erb-B2 as compared to the EGF-dependent subline (S1). Moreover, by application of a blocking anti-EGF-R antibody, growth of S2 cells in EGF-free medium was inhibited significantly, indicating that EGF-R was involved in an autocrine loop probably with transforming growth factor-alpha as ligand. Neither the late passages of S1 cells nor S2 cells were tumorigenic after subcutaneous transplantation to athymic mice. Our results indicate that c-myc amplification and overexpression are correlated with a decreased requirement for growth factors. Even when these alterations are combined with immortalization and EGF independence, they are insufficient for malignant transformation of these human breast epithelial cells.

Estrogen receptor messenger RNA splice variants are not involved in antiestrogen resistance in sublines of MCF-7 human breast cancer cells.

Development of resistance to tamoxifen is a serious problem in treatment of breast cancer patients. Although the mechanisms for development of resistance are unclear, an altered expression of alternatively spliced estrogen receptor (ER) mRNA has been suggested to be involved. We have looked for differential expression of ER splice variants lacking exon 2 (ERdeltaE2), exon 3 (ERdeltaE3), exon 4 (ERdeltaE4), exon 5 (ERdeltaE5), exon 7 (ERdeltaE7), and exons 4 and 7 (ERdeltaE4, 7) in the human breast cancer cell line MCF-7 and 10 ER-positive MCF-7 sublines resistant to the antiestrogens tamoxifen, ICI 164,384 or ICI 182,780. No major differences in the expression were demonstrated between MCF-7 cells and resistant cells, indicating that ER splice variants are not involved in antiestrogen resistance in this model system. Furthermore, despite a high mRNA level of some of the ER splice variants, no corresponding proteins could be detected using Western blot analysis.

The complex between urokinase and its type-1 inhibitor in primary breast cancer: relation to survival.

We examined the relationship between tumor tissue level of the complex formed of urokinase (uPA) and its type-1 inhibitor (PAI-1) and survival of breast cancer patients. The study included 342 axillary lymph node-negative and -positive primary breast cancer patients with a median follow-up of 67 months. Using a newly established ELISA, the levels of preformed uPA-PAI-1 complex were measured in tumor tissue extracts and analyzed with respect to total uPA, total PAI-1, and clinicopathological parameters, including survival. uPA-PAI-1 complex comprised a minor, variable fraction of both total uPA and PAI-1 levels. The complex levels were higher in node-negative tumors than in node-positive tumors and higher in small and low-grade tumors (all, P < or = 0.002). The tumor levels of complex, uPA, and PAI-1 were all associated with survival; high complex levels predicted longer recurrence-free survival (P = 0.03) and overall survival [OS (P = 0.005)], whereas high uPA or PAI-1 levels significantly predicted shorter survival. In multivariate Cox analysis, the only parameters that independently predicted survival were total PAI-1 level and lymph node status for recurrence-free survival and OS and, additionally, steroid hormone receptor status and grade for OS. This is the first demonstration of a relationship between uPA.PAI-1 complex tumor level and patient survival. However, total PAI-1 level showed superior prognostic power. Additional studies are needed to understand the relationship of these parameters to cancer biology and to assess the clinical utility of the uPA PAI-1 complex.