The surface of prostate carcinoma DU145 cells mediates the inhibition of urokinase-type plasminogen activator by maspin.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.

Linking gene expression patterns to therapeutic groups in breast cancer.

A major objective of current cancer research is to develop a detailed molecular characterization of tumor cells and tissues that is linked to clinical information. Toward this end, we have identified approximately one-quarter of all genes that were aberrantly expressed in a breast cancer cell line using differential display. The cancer cells lost the expression of many genes involved in cell adhesion, communication, and maintenance of cell shape, while they gained the expression of many synthetic and metabolic enzymes important for cell proliferation. High-density, membrane-based hybridization arrays were used to study mRNA expression patterns of these genes in cultured cells and archived tumor tissue. Cluster analysis was then used to identify groups of genes, the expression patterns of which correlated with clinical information. Two clusters of genes, represented by p53 and maspin, had expression patterns that strongly associated with estrogen receptor status. A third cluster that included HSP-90 tended to be associated with clinical tumor stage, whereas a forth cluster that included keratin 14 tended to be associated with tumor size. Expression levels of these clinically relevant gene clusters allowed breast tumors to be grouped into distinct categories. Gene expression fingerprints that include these four gene clusters have the potential to improve prognostic accuracy and therapeutic outcomes for breast cancer patients.

Reduced mammary tumor progression in WAP-TAg/WAP-maspin bitransgenic mice.

Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration.

Maspin--a novel protease inhibitor with tumor-suppressing activity in breast cancer.

Maspin (mammary serpin) is a novel serine protease inhibitor related to the serpin family with a tumor-suppressing function in breast cancer. Maspin was originally identified from normal mammary epithelium by subtractive hybridization and might function as a class II tumor-suppressor gene. Maspin's decreased expression with increased level of malignancy and its loss in metastatic cells is regulated at the transcriptional level. Cytosin methylation and heterochromatinization in the promoter region might account for this down-regulation of maspin. Transfection of tumor cells with maspin cDNA inhibits invasion and motility and decreases tumor growth and metastatic ability in nude mice. Maspin interacts with the p53 tumor-suppressor pathway and function as an inhibitor of angiogenesis in vitro and in vivo. The progressive loss of expression of maspin during tumor progression makes this new protein an interesting diagnostic and prognostic marker. The re-expression of maspin by pharmacological intervention potentially offers a promising approach as a therapeutic option in breast cancer therapy.

Maspin plays an important role in mammary gland development.

Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.

Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter.

BACKGROUND: Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation. MATERIALS AND METHODS: The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. RESULTS: The mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the -619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream cis-acting promoter elements were identified. Our data indicate that the AP-1 element located between -139 and -133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 35-bp element located from -178 to -139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. CONCLUSIONS: Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.

maspin suppresses the invasive phenotype of human breast carcinoma.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

Identification and verification of differential display cDNAs using gene-specific primers and hybridization arrays.

An accurate and streamlined approach to differential display (DD) band identification and verification is described. To minimize false positives, the strategy avoids the use of impure Northern blot probes obtained from PCR-amplified DD bands. To increase throughput, the cloning of DD bands is replaced by a gene-specific primer approach, and hybridization arrays are used in place of Northern blots. In summary, DD bands obtained with long primers were directly sequenced to allow the design and synthesis of gene-specific primers, which were then used to PCR-amplify homogeneous probes for the verification of expression patterns by hybridization array analysis. Differential expression of 60 of the 63 genes tested was confirmed. Thus, false positives are not inherent to DD. The results demonstrate the power of DD used with hybridization arrays to rapidly generate information on expression patterns of differentially expressed genes.

Expression genetics: a different approach to cancer diagnosis and prognosis.

Expression genetics is a new approach to the identification of cancer-related genes. Instead of studying gene mutations at the genome level, it focuses on the investigation of heredity at the RNA level. By isolating genes whose expression is up or down regulated in cancers, expression geneticists study their function in the context of gene regulation. A major goal of expression genetics in cancer is to correct gene expression in tumors by the application of potential therapeutic agents.

Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin.

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion in vitro and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.

Protein transport across the in vitro perfused human placenta.

PROBLEM: Placental transport of various proteins present in human serum, such as immunoglobulins (IgG, IgA), specific anti-tetanus IgG (anti-TT-IgG), and tetanus toxoid-antigen (TT-AG), was investigated. In addition, the transport of IgG modified with biotin (IgG-BT) and 14C-bovine serum albumin (14C-BSA, a permeability marker for macromolecules), was assessed. METHOD OF STUDY: During the perfusion of an isolated cotyledon from human term placenta the perfusate was recirculated on both maternal and fetal sides. After an initial stabilisation phase of 2 hr (control phase), media on both sides were exchanged and perfusion was continued comparing two different conditions (experimental phase). In the first group (control experiments [A, n = 3]), no test proteins were added during the experimental phase (4-6 hr). In the second group (B, n = 5), during the experimental phase (6 hr) the maternal perfusion medium contained IgG (Sandoglobuline, 6-10 g/L), anti-TT-IgG (21-25 mg/L), TT-AG (0.19-0.24 mg/L), and IgA (0.13-0.19 g/L). IgG-BT (2 g/L) and 14C-BSA (30-40 nCi/ml) were added to the medium on the maternal side. IgGs and TT-AG were determined by specific enzyme-linked immunosorbent assay. RESULTS: Both groups showed stable metabolic conditions with constant rates of glucose consumption, lactate production, and hormone (human chorionic gonadotropin, human placental lactogen) release observed throughout the experiment. Washout levels of endogenous IgG and IgA observed in the maternal circuit at the end of the control period were 5 and 1000 times higher than in the fetal circuit. In the experimental phase these levels remained constant at 50-80% of control levels with no change in the last 4 hr of perfusion (group A). In group B, with addition of extra proteins, trace amounts of IgG-BT, IgA, and 14C-BSA were detectable in the fetal circuit within 1 hr, with no significant further increase in circulating levels in the following 4 hr of the perfusion. In contrast, the detection of IgGs in the fetal circuit was delayed by 2 hr; thereafter, a continuous linear increase was observed for all IgGs. TT-AG in fetal perfusate was below the detection limit. TT-AG was found on the fetal side only after ultrafiltration of samples obtained at the end of the experiment. For permeability comparison, the ratio between concentrations on the fetal and maternal side multiplied by 100 ([F:M] x 100), as detected after 6 hr of perfusion, was assessed (n = 5, mean +/- SD). Labelling of IgG with biotin (IgG-BT) reduced its placental transfer by a factor 10 (0.04 +/- 0.01) when compared with the natural IgG (0.49 +/- 0.08) or the specific antibody (anti-TT-IgG). The relative fetal-to-maternal ratio found for TT-AG (0.48 +/- 0.12) was similar to anti-TT-IgG (0.46 +/- 0.11), and approximately 4 and 50 times that of 14C-BSA (0.12 +/- 0.03) and IgA (0.01 +/- 0.01), respectively. Considering that the molecular weights of TT-AG and anti-TT-IgG were at least twice that of BSA and similar to IgA, the difference in transfer suggests a specific mechanism of transport. CONCLUSIONS: Compared with other proteins there is a significantly increased transfer of IgGs across the in vitro perfused human placenta from the maternal to the fetal side, indicating a specific transport mechanism. The similarity in transfer of anti-TT-IgG and tetanus antigen may suggest the transport as antibody-antigen complex.

Re-expression of elafin in 21MT2 breast carcinomas by phorbol 12-myristate 13-acetate is mediated by the Ap1 site in the elafin promoter.

elafin is a unique elastase inhibitor. It is differentially expressed at the transcriptional level in human normal mammary epithelial cells and carcinomas. The elafin gene is induced by PMA in 21MT2 breast tumor cells. By deletion analysis and mutagenesis, we have identified the Ap1 site in the promoter as the cis element mediating transcriptional activation of elafin in 70N normal breast cells and its induction by phorbol 12-myristate 13-acetate (PMA) in 21MT2 breast tumors. PMA treatment induces AP1 factor expression, which binds to the Ap1 site of the elafin promoter. Mutation of this Ap1 abolishes the capability of induction by PMA. Furthermore, our data provide a basis for therapeutic manipulation of proteinase inhibitors such as elafin.

Expression of maspin in prostate cells is regulated by a positive ets element and a negative hormonal responsive element site recognized by androgen receptor.

Prostate cancer is the most common cancer in men. The molecular mechanisms leading to its development are poorly understood. Maspin is a tumor-suppressing serpin expressed in normal breast and prostate epithelium. We have found that expression of maspin in normal and carcinoma-derived prostate epithelial cells is differentially regulated at the transcriptional level. We have identified two different kinds of cis elements, Ets and hormonal responsive element (HRE), in the maspin promoter. The Ets element is active in regulating maspin expression in normal prostate epithelial cells but inactive in tumor cells. The HRE site is a negative element that is active in both cell types. This negative DNA sequence can repress a heterologous promoter recognized by the androgen receptor. We conclude that expression of maspin is under the influence of both a positive Ets and a negative HRE element. Loss of maspin expression during tumor progression apparently results from both the absence of transactivation through the Ets element and the presence of transcription repression through the negative HRE element recognized by androgen receptor.

Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin.

Tumor invasion and metastasis are processes poorly understood at the molecular level. Maspin is a serine protease inhibitor (serpin) with tumor-suppressing function in the mammary gland. Maspin gene expression is decreased with malignancy and is lost in metastatic cells. We show in this report that differential expression of maspin in normal and carcinoma-derived mammary epithelial cells is regulated at the transcriptional level. We have identified the Ets and Ap1 sites in the maspin promoter that are active in regulating maspin expression in normal mammary epithelial cells but inactive in tumor cells. The Ets site alone is sufficient to activate transcription in a heterologous promoter, whereas the Ap1 site cooperates with Ets in activation. The enhancing function by Ets and Ap1 elements is decreased in primary tumor cells (21NT) and is abolished in invasive tumor cells (MDA-231). Thus, loss of maspin expression during tumor progression results at least in part from the absence of transactivation through the Ets and Ap1 sites.

Expression genetics in cancer: shifting the focus from DNA to RNA.

Expression genetics is a conceptually different approach to the identification of cancer-related genes than the search for mutations at the genome level. While mutations lie at the heart of cancer, at least in its early stages, what is recognized here are phenotypic changes usually many steps removed from the initiating mutation. Classically cancer geneticists have concentrated on genomic changes and have ignored the productive potential of examining downstream events based on screening for differential gene expression between tumor cells and well matched normal counterparts. Genes involved in cancer affect the normal functions of many cellular processes: not only proliferation but cell-cell and cell-matrix interactions, DNA repair, invasion and motility, angiogenesis, senescence, apoptosis, and others. Yet very few cancer-related genes affecting these processes have been identified in human cancers by classical methods to find mutated genes despite enormous efforts. I report here our success in readily isolating more than 100 candidate tumor suppressor genes from human tissue, estimated to represent roughly 20% of the total genes recoverable by this approach. Half of the genes are unknown and the other half include representatives of most known cancer processes. Because their expression is lost during cancer progression, they may be useful tumor markers for diagnosis and prognosis. Because these genes are not mutated, they provide opportunities for pharmacological intervention by inducing their reexpression.

Maspin. A tumor suppressing serpin.

Maspin, a serpin found in mammary epithelial cells, has been shown to have tumor suppressor activity. The gene is expressed in normal human mammary epithelial cells but down-regulated in invasive breast carcinomas. Similar patterns of expression at the RNA and protein levels are seen by Northern analysis with cells grown in culture and by immunostaining of tissues. Biological assays of invasion by tumor cells through Matrigel membranes and of motility have shown that recombinant maspin inhibits both processes, and that its inhibitory action is totally lost by a single cleavage at the reaction center. Tumor transfectants expressing maspin are inhibited in growth and metastasis in nude mice. Maspin is located in the cell membrane and extracellular matrix, and does not behave as a classical inhibitory serpin against any known target protease. Its mode of action is presently unknown.

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.