Basement Membrane Protein Laminin Research Articles

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.

Expression of Basement Membrane Proteins: Evidence for Complex Post-transcriptional Control Mechanisms

Differentiated murine teratocarcinoma cell lines have been widely used as sources for the basement membrane proteins laminin and collagen IV. In order to understand the control of their expression, we have measured the transcription rates of the corresponding genes in nuclear run-on assays. The ratios of transcripts obtained from the five different genes of interest (for laminins A, B1, and B2 and α1(IV), α2(IV)) are rather different from the ratios of the corresponding mRNAs, which are again different from the protein levels needed. The gene for α2(IV) is transcribed at a higher rate than the one for α1(IV) and, similarly, the gene for laminin A is transcribed at a higher rate than the other two laminin genes, respectively. However, the α2(IV) and laminin A mRNA levels are lower than those for the other chains of the same molecule. The α1(IV) mRNA is 3- to 15-fold more abundant than the α2(IV) mRNA, depending on the cell line. At the protein level, the A chain seems to be limiting for the assembly of laminin, in accordance with its low mRNA level. The two collagen chains have variable pool sizes, but the triple helical molecules always seem to be composed of two α1(IV) and one α2(IV) chains. These results point to extensive control mechanisms at various stages of post-transcriptional events, some of which we could identify.

Analysis of time-varying imaging from phase-contrast microscopy

Quantitative analysis of neurite outgrowth and growth cone (filopodia and lamellipodia) motility provides key information for developing new therapeutic strategies for treating peripheral nerve injury and disease. Neurite outgrowth and growth cone properties will be measured for neurons taken from the dorsal root ganglia of 8-day old chick embryos. Ganglia will be enzymatically dissociated into single neurons and cultured in a serum-free medium. Cultures will be grown on glass coverslips coated with the basement membrane protein laminin. Following a brief incubation period of 4 hr to allow the initiation of neurites from the nerve cell bodies, cultures will be moved to an incubated microscope stage maintained at 37 C. Subsequent growth of the neurites will be recorded at a rate of 2 frames/sec with a time-lapse videocassette recorder connected to the phase-contrast microscope through a high-resolution (800 lines) video camera.The videotaped growth sequence will be digitized at a specified time interval, using a frame grabber.

Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells.

Polarized epithelial cells target distinct sets of membrane and secretory proteins to their apical and basolateral domains. Here we examine whether constitutively secreted and membrane proteins that are bound for the same domain share the same carrier vesicles. To address the issue, differential effects of microtubule depolymerization on basolateral protein targeting in the polarized Madin-Darby canine kidney II cell line were studied. We find that the basolateral insertion of the active, ouabain-binding Na+,K(+)-ATPase and of a set of very late antigen integrins is little affected by microtubule disruption. Under equivalent conditions, the basolateral secretion of the basement membrane protein laminin is strongly suppressed. More specifically, it is demonstrated that microtubules are involved in targeting laminin, but not integrins, from the compartment related to the accumulation of newly synthesized proteins at 20 degrees C (trans-Golgi network) to the basolateral domain. Our study also reveals that laminin associated with basolateral binding sites interacts with those sites only secondarily to secretion. The data provide evidence for a branch in the basolateral targeting pathway, with secreted and membrane proteins loaded into distinct carrier vesicles.

Thromboxane stimulates synthesis of extracellular matrix proteins in vitro

The vasoconstrictor eicosanoid thromboxane plays an important role in the pathogenesis of several renal diseases. As an autacoid, its local release alters blood flow and induces platelet aggregation. We report a direct stimulatory effect of thromboxane on extracellular matrix protein production and gene expression in vitro. Treatment of two cell types, differentiated mouse teratocarcinoma cells (F9+) and human glomerular mesangial cells, with two different thromboxane analogues resulted in increased production of components of the extracellular matrix including fibronectin and the basement membrane proteins laminin and type IV collagen. These responses to thromboxane were not the result of a mitogenic effect of thromboxane nor the result of an increase in total cellular protein. The increased production of extracellular matrix proteins was, at least in part, due to an increase in the steady-state level of mRNA for these genes. Furthermore, the effect of thromboxane was markedly inhibited by cotreatment with a thromboxane-receptor antagonist. These results suggest a new potential role for thromboxane as a mediator of the sclerotic and fibrotic responses to injury.

Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits.

The distribution of subunits of the basement membrane proteins laminin and merosin in human and rabbit tissue was studied by immunofluorescence using monoclonal antibodies. The laminin A chain is present in epithelial, endothelial, and smooth muscle basement membranes. Merosin, as defined by its heavy chain M, is present in striated muscle and peripheral nerve. The A subunit colocalizes with at least two B subunits: B2 plus either B1 or the recently discovered B1 homologue S. The M subunit most often colocalizes with B1 and B2. Exceptions include the myotendinous junction, where M colocalizes with S, and the trophoblast basement membrane, where the M subunit colocalizes with S as well as B1. The presence of all five known subunits of the laminin family in placenta allowed isolation of their parent molecules in native form by the use of monoclonal antibodies in affinity chromatography. Four different heterotrimeric proteins could be identified: B1 chain-containing laminin (A-B1-B2), S chain-containing laminin (A-S-B2), B1-containing merosin (M-B1-B2), and S-containing merosin (M-S-B2). The data show that the proteins in the laminin family are heterotrimers composed of one heavy and two light chains; that most basement membranes contain predominantly one protein of the laminin family; and that laminin, as defined by the A subunit, has a much more restricted distribution than previously thought.

Binding and immunological properties of a synthetic peptide corresponding to the phosphorylcholine-binding region of C-reactive protein

A synthetic peptide corresponding to amino acid residues 47–63 of human C-reactive protein (CRP) was synthesized and evaluated for its ability to bind phosphorylcholine (PC) and to react with mAb specific for the PC-binding region of CRP. The PC-binding peptide displayed Ca 2+-independent binding specific for PC and was able to compete against CRP for PC in the presence of Ca 2+ ions. The synthetic peptide, like CRP, binds to the extracellular matrix protein fibronectin and the basement membrane protein laminin. The PC-binding peptide was recognized by those mAb generated against the intact CRP molecule that bind at, or near, the functional PC-binding region. In addition, several mAb to the T-15 idiotype present on mouse antibodies specific for PC, recognize an epitope(s) on the PC-binding peptide. Therefore, the 17 amino acid synthetic peptide shares both functional binding activity and antigenicity with the corresponding functional region within the CRP molecule.

Changes Associated With Metastasis in B16-F1 Melanoma Cells Surviving Heat

Metastasis to distant sites is mediated by various receptors on the surface of tumor cells. B16-F1 melanomas surviving 43.5 degrees C heat in vitro for 15 minutes and cultured for 10 days bind significantly increased amounts of the basement membrane protein laminin. Motility of heat-resistant B16-F1 cells in vitro toward the chemoattractant laminin is significantly increased. The increased expression of putative laminin receptors may be associated with increased metastasis of melanomas after subcurative hyperthermia.

Characteristics of the binding of human C‐reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.

Immunoelectron microscopic localization of laminin, type IV collagen, and type III pN-collagen in reticular fibers of human lymph nodes.

We studied the ultrastructural distribution of laminin, type IV collagen, and the amino terminal pro-peptide of type III collagen (type III pN-collagen) in normal human lymph nodes. After fixation with freshly prepared 4% paraformaldehyde mixed with 0.1% glutaraldehyde, cryoultramicrotomy proved to preserve the antigenicity of these proteins better than embedding in Lowicryl K4M. Sections were treated with rabbit antibodies against the 7S domain of human type IV collagen, the fragment P1 of human laminin, and the amino terminal pro-peptide of human type III pro-collagen, followed by anti-rabbit IgG conjugated to 10-nm colloidal gold. Laminin and type IV collagen were seen in the basement membrane structures of the blood vessels and in the walls of sinuses. The amorphous material between the collagenous fibers in locations corresponding to reticular fibers also contained laminin and type IV collagen. The amino terminal pro-peptide of type III pro-collagen was present in the collagenous fibers in reticular fibers and in the walls of blood vessels and sinuses. Therefore, a significant number of the type III collagen molecules in these fibers must have retained their amino terminal pro-peptide. These results indicate that the basement membrane proteins laminin and type IV collagen are genuine components of reticular fibers, as suggested earlier by immunohistochemical studies at the light microscopic level.

Respiratory burst in adherent human neutrophils: triggering by colony- stimulating factors CSF-GM and CSF-G

Human neutrophils adherent to proteins derived from serum or plasma, or to the basement membrane protein laminin, underwent a delayed but massive respiratory burst in response to recombinant human CSF-GM or CSF-G. No such response was elicited from neutrophils in suspension. On a molar basis, CSF-GM (EC50 approximately 126 pmol/L) and CSF-G (EC50 approximately 585 pmol/L) were about as potent as TNF alpha and TNF beta in their elicitation of H2O2 release and orders of magnitude more potent than previously studied formylated peptides or C5a. CSF-GM and CSF-G prime suspended neutrophils for a respiratory burst in response to soluble agonists, such as formylated peptides. Compared to the CSF-primed respiratory burst of nonadherent neutrophils, the CSF-triggered response of adherent neutrophils is markedly more delayed in onset (73 to 95 minutes), prolonged in duration (150 to 180 minutes), and greater in extent (approximately 60 to 100 nmol H2O2 released/10(6) neutrophils). Neither CSF-M, interleukin-3 (IL-3), nor bacterial lipopolysaccharide triggered the respiratory burst in adherent neutrophils, nor did CSF-GM or CSF-G trigger a respiratory burst in adherent monocytes. Release of CSF-GM and CSF-G in response to antigens, bacterial products, or cytokines may give mononuclear cells control over the respiratory burst of noncirculating neutrophils during inflammatory and immune responses.

Respiratory Burst in Adherent Human Neutrophils: Triggering by Colony-Stimulating Factors CSF-GM and CSF-G

Abstract Human neutrophils adherent to proteins derived from serum or plasma, or to the basement membrane protein laminin, underwent a delayed but massive respiratory burst in response to recombinant human CSF-GM or CSF-G. No such response was elicited from neutrophils in suspension. On a molar basis, CSF-GM (EC50 approximately 126 pmol/L) and CSF-G (EC50 approximately 585 pmol/L) were about as potent as TNF alpha and TNF beta in their elicitation of H2O2 release and orders of magnitude more potent than previously studied formylated peptides or C5a. CSF-GM and CSF-G prime suspended neutrophils for a respiratory burst in response to soluble agonists, such as formylated peptides. Compared to the CSF- primed respiratory burst of nonadherent neutrophils, the CSF-triggered response of adherent neutrophils is markedly more delayed in onset (73 to 95 minutes), prolonged in duration (150 to 180 minutes), and greater in extent (approximately 60 to 100 nmol H2O2 released/10(6) neutrophils). Neither CSF-M, interleukin-3 (IL-3), nor bacterial lipopolysaccharide triggered the respiratory burst in adherent neutrophils, nor did CSF-GM or CSF-G trigger a respiratory burst in adherent monocytes. Release of CSF-GM and CSF-G in response to antigens, bacterial products, or cytokines may give mononuclear cells control over the respiratory burst of noncirculating neutrophils during inflammatory and immune responses.

Characterization of Extracellular Matrix in Non-Hodgkin's Lymphomas

Basement membrane proteins laminin, type IV collagen and aminoterminal propeptide of type III collagen (type III pN-collagen) are important components of extracellular matrix of normal lymph nodes; they are present in reticular fibres and in the walls of blood vessels and sinuses. We have studied the distribution of these proteins in 45 cases of non-Hodgkin's lymphomas representing different types of Kiel classification. Light microscopic immunohistochemistry was used on sections from formalin fixed, paraffin embedded biopsy specimens. Reticular cells were identified by using antibodies against S-100 protein, a marker for dendritic and interdigitating reticulum cells, and against cystatin A for the demonstration of follicular dendritic reticulum cells. The findings of the immunohistochemical stainings were generally similar in the different lymphoma types. Thus the method for the demonstration of extracellular matrix proteins could not give obvious aid for lymphoma classification. BM stainings proved to be efficient in the demonstration of sinuses. Marginal and medullary sinuses of the nodal cases were either totally or partly spared and open also in the cases in which there was extracapsular growth. The fibre content was variable, but generally the distribution of all the three extracellular matrix proteins was comparable with each other and mainly corresponded with the pattern seen in conventional Gomori's reticulin stain. This also holds for the tumor tissue infiltrated through the capsule and for extranodal cases. Areas of sclerosis of some cases contained only type III pN-collagen. Reticular fibres were at places seen to be closely associated with cells of spindle or dendritic shape. A few reticulum cells could be found to show cytoplasmic staining for laminin, type IV collagen and type III pN collagen. This finding suggests that the cells are synthetizing these proteins. The amount of cells staining for cystatin A or S-100 protein did not generally correlate with the amount of fibres, and most of the stained cells were not closely associated with fibres. The results show that in non-Hodgkin's lymphomas basement membrane proteins and type III pN-collagen are important components of reticular fibres. Results also indirectly support the main role of fibroblastic reticulum cells in the production of the fibres.

The N terminus of laminin A chain is homologous to the B chains.

A major proteolytic fragment (E1/E1-4) of the basement membrane protein laminin, comprising the three short arms with some terminal globules missing, was isolated by elastase digestion, and partial protein sequence data were determined for several tryptic peptides. Sequences which corresponded to A-chain structures were used to synthesize oligonucleotides for the construction and screening of a primer-extended cDNA library from mouse PYS-2 cells. A clone of 1.1 kb was obtained and shown by sequencing to correspond to the 5' end of the 10-kb mRNA of the A chain of laminin. The clone contains 77 nucleotides of 5' untranslated sequence and a region coding for 334 amino acids, including a presumptive signal peptide of 24 amino acids. The sequence is 30% homologous to the corresponding N-terminal part of the B1 chain of laminin, suggesting the same structure for both domains. The data present further evidence for a recent structural model which postulates that each of the three laminin polypeptide chains forms a distinct short arm.

Fibronectin Extracellular Matix Assembly by Human Epidermal Cells Implanted Into Athymic Mice

Human epidermal keratinocytes reorganize into epidermal inclusion cysts when implanted subcutaneously into athymic mice. During the organization and maturation of these cysts, fibronectin accumulates in the surrounding extracellular matrix and the basement membrane proteins bullous pemphigoid antigen and laminin appear at the epithelial-stromal interface. The sequence in which these proteins appear parallels that seen during reepithelialization of a skin wound in vivo. Fibronectin appears during aggregation of the epidermal cells and persists in the area surrounding the cysts for at least 7 days. Bullous pemphigoid antigen and laminin appear later (by 4 and 7 days, respectively) and ultimately become organized into a continuous band at the periphery of the cyst. This distribution of bullous pemphigoid antigen and laminin at the stromal-epithelial interface persists at least 5 weeks, suggesting that the implanted epidermal cells are capable of developing and maintaining a stable basement membrane zone. Fibronectin, which is abundant in the matrix adjacent to the epidermal cysts and in the surrounding stroma during cyst organization and maturation, diminishes to undetectable levels by 5 weeks. While much of the fibronectin derives from the host tissues, species-specific antibodies to human fibronectin reveal that at least a portion of this protein is synthesized and deposited by the implanted epidermal cells.

Stem cells in mammary gland differentiation and cancer

ABSTRACT Evidence based on ultrastructure and immunocytochemical staining suggests that morphological gradations between epithelial and myoepithelial cells, and possibly between epithelial and alveolar-like cells, can occur in terminal ductal structures of rat and human mammary glands. In neoplastic disease the benign, carcinogen-induced rat and benign, human mammary tumours can contain epithelial, myoepithelial-like and alveolar-like cells, whereas their malignant counterparts mainly contain only epithelial-like cells. Clonal epithelial cell lines from normal rat mammary glands, from benign tumours and from SV4O-transformed human mammary cultures can differentiate to either myoepithelial-like or alveolar-like cells. In those of the rat, the differentiation processes occur in steps, intermediate cells along the myoepithelial-like pathway resemble the morphological intermediates in the terminal ductal structures in vivo. Changes in specific polypeptides characterize each of the intermediate cells in vitro. One of the earliest increases observed in the myoepithelial-like pathway in vitro is that due to a novel protein p9Ka, whereas the major increases in Thy-1 antigen and the basement membrane proteins laminin and type IV collagen occur at later steps. The nucleotide sequence of the gene for p9Ka is related to that of the small, regulatory calcium-binding proteins, and antibodies raised to synthetic fragments of its predicted amino acid sequence react with only myoepithelial cells within the rat mammary parenchyma. Increases in the production of p9Ka and Thy-1 are largely due to increases in their messenger RNAs, possibly arising at the level of transcription of the DNA, whereas the increases in production of laminin and type IV collagen occur at a post-transcriptional level. The normal transcriptional promoter sequences of TATA or CAAT are not found adjacent to the genes for p9Ka or Thy-1. Cells and cell lines from malignant rat mammary tumours of increasing metastatic potential and from malignant areas of human ductal carcinomas largely fail to yield fully differentiated myoepithelial-like or alveolar-like cells in culture; however, weakly metastasizing rat cells yield variants which may retain a vestige of the myoepithelial phenotype. It is suggested that novel regulatory transcriptional element(s) may control the production of some of the proteins along the normal myoepithelial-like pathway, and that these elements may be relatively unique in their capacity to become inoperative in the malignant breast cancer cell.

37] Assays of growth factor stimulation of extracellular matrix biosynthesis

The chapter presents some of the more useful methods for the quantitative and qualitative measurements of extracellular matrix components affected by growth factors. In primary culture a strong, positive correlation between the growth stimulating potencies of growth factors and their abilities to differentially amplify basement membrane protein–laminin, collagen IV, and proteoglycan, synthesis has been established. Additionally, when mammary cells are cultured on different types of extracellular matrix proteins, they display differing requirements for growth factors. These observations suggest that at least part of the mechanism by which growth factors act is by enhancing the biosynthesis of extracellular matrix proteins. This is especially true in the case of primary cell cultures in which cell selection for culture conditions has not taken place. Thus quantitation of growth factor requires analysis of cells and growth medium. Growth factors have been shown to alter both the biosynthetic and degradative rates of matrix proteins, especially of collagen, the component that has been studied in greatest detail.

Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor.

A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.

Serum concentrations of laminin and aminoterminal propeptide of type III procollagen in relation to the portal venous pressure of fibrotic liver diseases

In sera of patients with fibrotic liver diseases ( n = 33) classified histologically into various degrees of liver fibrosis ( n = 21) and cirrhosis ( n = 12) the concentrations of the basement membrane protein laminin and of its pepsinresistant fragment P1 and of the N-terminal propeptide of type III procollagen were determined. The concentrations of both proteins were related to the portal venous pressure measured in these patients. Compared with the reference population ( n = 146) the concentration of laminin increases from 1.04 U/ml (normal persons) to 1.69 ± 0.46 U/ml in liver fibrotic and 2.58 ± 0.87 U/ml in liver cirrhotic patients. Although the concentrations of the propeptide of type III procollagen increase also there exist only weak correlations between both connective tissue proteins in serum. Laminin is correlated highly positive with the portal venous pressure in cirrhotic subjects ( r = 0.9206), the extent of laminin elevation reflects closely the degree of portal hypertension. Virtually all of the fibrotic patients having a laminin concentration within the reference range had a normal portal venous pressure. The data suggest laminin as a potentially useful parameter for monitoring the portal venous pressure in cirrhotic and severe fibrotic patients.

Characterization of rat mammary cell types in primary culture: Lectins and antisera to basement membrane and intermediate filament proteins as indicators of cellular heterogeneity

Three morphologically distinct major cell types were observed in primary cultures obtained from the mammary parenchyma of glands from virgin rats. These cell types consisted of small cuboidal epithelial cells, larger epithelioid cells and elongated cells. We have investigated the distribution of the basement membrane proteins laminin and type IV collagen, and the intermediate filament proteins vimentin and prekeratin, in these three cell types using immunofluorescence techniques. Antisera to the basement membrane proteins stain the large epithelioid cells and the elongated cells, but do not stain the small cuboidal cells. Polyclonal antiserum to keratin stains all the small cuboidal and large epithelioid cells, but only a small subpopulation of the elongated cells. However, a monoclonal antibody to keratin, LP34, stains only the large cuboidal and a proportion of the elongated cells. Vimentin antiserum fails to stain the small cuboidal cells but stains all the large epithelioid and elongated cells. In addition, peanut lectin, which binds only to ductal lining epithelial cells in the virgin rat mammary gland in vivo after their treatment with neuraminidase, binds to the small cuboidal cells after neuraminidase treatment but not to the other cell types. However, Griffonia simplicifolia agglutinin I, which specifically stains myoepithelial cells in vivo, binds to the large epithelioid and elongated cells but not to the small cuboidal cells. These results suggest that the small cuboidal cells are related to mammary ductal epithelial cells whereas the large epithelial and elongated cells have some characteristics of myoepithelial cells.