An OLTAM system for analysis of brown/beige fat thermogenic activity.

BACKGROUND/OBJECTIVES: Thermogenic fat is present in humans and emerging evidence indicates that increasing the content and activity of these adipocytes may lead to weight loss and improved metabolic health. Multiple reporter systems have been developed to assay thermogenic fat activity based on the transcriptional and translational activation of Ucp1, the key molecule that mediates nonshivering thermogenesis. Our study aims to develop a much-needed tool to monitor thermogenic fat activity through a mechanism independent of Ucp1 regulation, therefore effectively assaying not only canonical ß-adrenergic activation but also various non-UCP1-mediated thermogenic pathways that have been increasingly appreciated. METHODS: We detected increased luciferase activity upon thermogenic activation in interscapular brown and inguinal subcutaneous fat in ODD-Luc mice, a hypoxia reporter mouse model. We then developed an OLTAM (ODD-Luc based Thermogenic Activity Measurement) system to assay thermogenic fat cell activity. RESULTS: In both primary murine and human adipocytes and an immortalized adipose cell line that were transduced with the OLTAM system, luciferase activity can be readily measured and visualized by bioluminescence imaging in response to a variety of stimuli, including UCP1-independent thermogenic signaling. This system can offer a convenient method to assay thermogenic activity for both basic and translational research. CONCLUSIONS: The OLTAM system offers a convenient way to measure the activation of thermogenic fat and presents opportunities to discover novel signaling pathways and unknown compounds targeting metabolically active adipocytes to counteract human obesity.

Molecular complexity of leukocyte surface glycoproteins related to the macrophage differentiation antigen Mac-1.

Monoclonal antibodies have been obtained that react with the murine leukocyte surface glycoproteins bearing the macrophage differentiation antigen Mac-1. Structural and antigenic analysis shows that related glycoproteins are found on other murine hematopoietic cell types. The data not only illustrate the complexity of cell surface structures that can be detected by means of monoclonal antibodies, but also raise questions as to the functional significance of this family of molecules within the hematopoietic system.

Human homologue of murine T200 glycoprotein.

We report the identification of the human homologue of murine T200 glycoprotein. Peptide-mapping experiments suggest that the structure of the glycoprotein is highly conserved between the two species. Many of the properties of human T200 homologue are similar to those of murine T200 glycoprotein: it is broadly distributed within the hematopoietic system but is not detectable on nonhematopoietic cells; there are also structural differences between the forms of the glycoprotein found on T and B lymphoblastoid cell lines. These results suggest the homologous glycoproteins may play similar functional roles in both species.

T200 cell surface glycoprotein of the mouse. Polymorphism defined by the Ly-5 system of alloantigens.

The cell line BW5147, and a mutant T200-negative cell line derived from BW5147, were studed by immunoprecipitation and peptide mapping, with xenogeneic monoclonal anti-T200 serum and with Ly-5 alloantiserum. It appears that the Ly-5 system defines a structural polymorphism of the cell surface glycoprotein T200, and that the monoclonal anti-T200 serum defines a feature of T200 that is common to mice of both Ly-5a and Ly-5b genotypes and may be invariable in the species.

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice.

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-alpha/beta and IFN-gamma receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-alpha/beta receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-gamma receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor.

14-3-3 is a ubiquitous protein family that interacts with several signal transduction kinases. We show that 14-3-3 proteins associate with keratin intermediate filament polypeptides 8 and 18 (K8/18) that are expressed in simple-type epithelia. The association is stoichiometrically significant (> or = one 14-3-3 molecule/keratin tetramer), occurs preferentially with K18, and is phosphorylation- and cell cycle-dependent in that it occurs during S/G2/M phases of the cell cycle when keratins become hyperphosphorylated. Binding of phospho-K8/18 to 14-3-3 can be reconstituted in vitro using recombinant 14-3-3 or using total cellular cytosol. Phosphatase treatment results in dissociation of 14-3-3, and dephosphorylation of phospho-K8/18 prevents reconstitution of the binding. Three cellular keratin subpopulations were analyzed that showed parallel gradients of keratin phosphorylation and 14-3-3 binding. Incubation of 14-3-3 with keratins during or after in vitro filament assembly results in sequestering of additional soluble keratin, only in cases when the keratins were hyperphosphorylated. Our results demonstrate a stoichiometrically significant cell cycle- and phosphorylation-regulated binding of 14-3-3 proteins to K18 and in vitro evidence of a simple epithelial keratin sequestering role for 14-3-3 proteins.

Keratins turn over by ubiquitination in a phosphorylation-modulated fashion.

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.

There is ample in vitro evidence that phosphorylation of intermediate filaments, including keratins, plays an important role in filament reorganization. In order to gain a better understanding of the function of intermediate filament phosphorylation, we sought to identify the major phosphorylation site of human keratin polypeptide 18 (K18) and study its role in filament assembly or reorganization. We generated a series of K18 ser-->ala mutations at potential phosphorylation sites, followed by expression in insect cells and comparison of the tryptic 32PO4-labeled patterns of the generated constructs. Using this approach, coupled with Edman degradation of the 32PO4-labeled tryptic peptides, and comparison with tryptic peptides analyzed after labeling normal human colonic tissues, we identified ser-52 as the major K18 physiologic phosphorylation site. Ser-52 in K18 is not glycosylated and matches consensus sequences for phosphorylation by CAM kinase, S6 kinase and protein kinase C, and all these kinases can phosphorylate K18 in vitro predominantly at that site. Expression of K18 ser-52-->ala mutant in mammalian cells showed minimal phosphorylation but no distinguishable difference in filament assembly when compared with wild-type K18. In contrast, the ser-52 mutation played a clear but nonexclusive role in filament reorganization, based on analysis of filament alterations in cells treated with okadaic acid or arrested at the G2/M stage of the cell cycle. Our results show that ser-52 is the major physiologic phosphorylation site of human K18 in interphase cells, and that its phosphorylation may play an in vivo role in filament reorganization.

Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues.

Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization.

Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant.

The two major intermediate filament proteins in glandular epithelia are keratin polypeptides 8 and 18 (K8/18). To evaluate the function and potential disease association of K18, we examined the effects of mutating a highly conserved arginine (arg89) of K18. Expression of K18 arg89-->his/cys and its normal K8 partner in cultured cells resulted in punctate staining as compared with the typical filaments obtained after expression of wild-type K8/18. Generation of transgenic mice expressing human K18 arg89-->cys resulted in marked disruption of liver and pancreas keratin filament networks. The most prominent histologic abnormalities were liver inflammation and necrosis that appeared at a young age in association with hepatocyte fragility and serum transaminase elevation. These effects were caused by the mutation since transgenic mice expressing wild-type human K18 showed a normal phenotype. A relative increase in the phosphorylation and glycosylation of detergent solubilized K8/18 was also noted in vitro and in transgenic animals that express mutant K18. Our results indicate that the highly conserved arg plays an important role in glandular keratin organization and tissue fragility as already described for epidermal keratins. Phosphorylation and glycosylation alterations in the arg mutant keratins may account for some of the potential changes in the cellular function of these proteins. Mice expressing mutant K18 provide a novel animal model for human chronic hepatitis, and for studying the tissue specific function(s) of K8/18.

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.

Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice.

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament (IF) proteins. One important physiologic function of K8/18 is to protect hepatocytes from drug-induced liver injury. Although the mechanism of this protection is unknown, marked K8/18 hyperphosphorylation occurs in association with a variety of cell stresses and during mitosis. This increase in keratin phosphorylation involves multiple sites including human K18 serine-(ser)52, which is a major K18 phosphorylation site. We studied the significance of keratin hyperphosphorylation and focused on K18 ser52 by generating transgenic mice that overexpress a human genomic K18 ser52--> ala mutant (S52A) and compared them with mice that overexpress, at similar levels, wild-type (WT) human K18. Abrogation of K18 ser52 phosphorylation did not affect filament organization after partial hepatectomy nor the ability of mouse livers to regenerate. However, exposure of S52A-expressing mice to the hepatotoxins, griseofulvin or microcystin, which are associated with K18 ser52 and other keratin phosphorylation changes, resulted in more dramatic hepatotoxicity as compared with WT K18-expressing mice. Our results demonstrate that K18 ser52 phosphorylation plays a physiologic role in protecting hepatocytes from stress-induced liver injury. Since hepatotoxins are associated with increased keratin phosphorylation at multiple sites, it is likely that unique sites aside from K18 ser52, and phosphorylation sites on other IF proteins, also participate in protection from cell stress.

Identification of nuclear receptors for VIP on a human colonic adenocarcinoma cell line.

Vasoactive intestinal peptide (VIP) is a neuropeptide with broad tissue distribution. Although its precise function is unknown, it is thought to exert its effect, at least in part, by interacting with cell surface receptors. Nuclear receptors for VIP have now been identified by specific binding of 125I-labeled VIP to nuclei of a human colonic adenocarcinoma cell line (HT29) and by cross-linking of 125I-labeled VIP to its receptor on intact nuclei. In contrast, 125I-labeled transferrin shows only background binding to nuclei but significant binding to intact cells. Purity of the isolated nuclei was further substantiated by electron microscopy. The apparent molecular sizes of the VIP--cross-linked nuclear and cell surface receptors are similar but not identical.

Dynamic effects of acid on Barrett's esophagus. An ex vivo proliferation and differentiation model.

Barrett's esophagus (BE), or specialized intestinal metaplasia, is a premalignant heterogeneous epithelium associated with reflux and an increased risk for adenocarcinoma. Since acid is a major component of refluxate, we investigated its effects ex vivo on cell differentiation as determined by villin expression; and on cell proliferation, as determined by tritiated thymidine incorporation and proliferating cell nuclear antigen expression. To mimic known physiological conditions, endoscopic biopsies of normal esophagus, BE, and duodenum were exposed, in organ culture, to acidified media (pH 3-5) either continuously, or as a 1-h pulse and compared with exposure to pH 7.4 for up to 24 h. Before culture, villin expression was noted in 25% of BE samples, and increased after 6 or 24 h of continuous acid to 50% or 83% of BE samples, respectively. Increased villin expression correlated with ultrastructural maturation of the brush border. In contrast, an acid-pulse followed by culture at pH 7.4, did not alter villin expression in BE. Moreover, continuous acid exposure blocked cell proliferation in BE, whereas, an acid-pulse enhanced cell proliferation, as compared to pH 7.4. Based on our ex vivo findings, we propose a model in which the diverse patterns of acid exposure in vivo may contribute to the observed heterogeneity and unpredictable progression to neoplasia of BE.

Mutation of human keratin 18 in association with cryptogenic cirrhosis.

Mutations in 11 of the more than 20 keratin intermediate filaments cause several epidermal and oral associated diseases. No disease-associated mutations have been described in keratin 8 or 18 (K8/18) which are the major keratin pair in simple-type epithelia, as found in the liver, pancreas, and intestine. However, transgenic mice that express mutant keratin 18 develop chronic hepatitis, and have an increased susceptibility to drug-induced hepatotoxicity. Also, ectopic expression of epidermal K14 in mouse liver results in chronic hepatitis, and disruption of mouse K8 leads to embryo lethality with extensive liver hemorrhage. We tested if patients with liver disease of unknown cause may harbor mutations in K18. We describe a his127-->leu (H127L) K18 mutation in a patient with cryptogenic cirrhosis that is germline transmitted. The K18 H127L isolated from the liver explant, or after expression in bacteria, showed an altered migration on two-dimensional gel analysis as compared with normal human liver or bacterially expressed K18. Electron microscopy of in vitro assembled K18 H127L and wild type K8 showed an assembly defect as compared with normal K8/18 assembly. Our results suggest that mutations in K18 may be predispose to, or result in cryptogenic cirrhosis in humans.

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant.

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.

Keratin modifications and solubility properties in epithelial cells and in vitro.

The gains that have been made in characterizing some of the keratin posttranslational modifications have helped answer some questions regarding these modifications and have generated an information base for asking additional refined questions in future studies. Highlights of where we believe we currently stand with regard to keratin posttranslational modifications are as follows: 1. Keratin glycosylation, via O-GlcNAc, is a dynamic modification that has been conclusively identified in K13, K8, and K18. Three serine glycosylation sites in the head domain of K18 have been identified, and it is possible that all keratins are glycosylated. The function of this modification remains to be defined, but is likely to be different from phosphorylation, since the two modifications are generally segregated on different molecules and several examples exist whereby both modifications increase simultaneously. 2. Keratin phosphorylation occurs within the tail and/or head domains of all keratins that have been examined. Several serine phosphorylation sites and some of the relevant kinases have been characterized in K8, K6, and K18, and serine/threonine sites have been identified in K1. Functions of keratin phosphorylation that have significant experimental support include a role in filament solubility and reorganization and a role in regulating keratin binding with other cytoplasmic proteins. The significance of filament reorganization and increased solubility under a variety of physiologic conditions such as mitosis and cell stress are important areas of future and ongoing investigation. Other associations with keratin phosphorylation include protection against cell stress, cell signaling, apoptosis, and cell compartment-specific roles. At this stage, however, it is not known if these associations play direct or indirect roles. 3. Keratin transglutamination occurs in epidermal and simple epithelial keratins under physiologic and pathologic states, respectively. In the physiological context, the role of this modification is clear in terms of providing a compact protective structure, while in the pathologic context of liver disease the role remains ambiguous. 4. Proteolysis of K18 and K19 by caspases occurs during apoptosis, and generates stable keratin fragments that are highly enriched within the cytoskeletal compartment. Proteolysis of the type II keratins appears to be spared for reasons that remain to be defined. It is likely that this apoptosis-associated degradation involves all type I keratins. Keratin fragments are also noted in sera of patients in association with a variety of epithelial tumors. If a signal does exist for the apoptosis-associated fragmentation, aside from caspase activation, then it appears that the overall increase in keratin phosphorylation during apoptosis does not account for this signal. 5. Keratins undergo several other posttranslational modifications including disulfide bond formation (not found in K8/18 due to lack of cystienes) and acetylation of their N-terminal serines. Modification by lipids is also possible, but this modification requires further confirmation. 6. Keratin solublility is highly dynamic and varies profoundly depending on the keratin pair and the physiologic state of the cell. Within the keratin family, simple epithelial keratins are among the most soluble (approximately 5% of K8/18 is soluble at basal conditions). Phosphorylation plays an important role in modulating keratin solubility, and distinct differences occur in site-specific phosphorylation depending on the soluble versus cytoskeletal partitioning of the keratin. Keratin solubility (at least for K8/18) also appears to be regulated by 14-3-3 proteins via K18 Ser33 phosphorylation.

A tyrosine sulfated human glycoprotein with an unusual cell distribution.

We describe a human integral cell surface glycoprotein (Mr 142 kD), recognized by monoclonal antibody M2B3. This glycoprotein is absent from resting peripheral blood lymphocytes, but becomes expressed in significant levels with mitogen activation. The M2B3 glycoprotein is present on epithelial cells in the basal layer of epidermal and esophageal tissue as well as in several fresh tumors examined. In addition, it is present on smooth muscle tissue throughout the gastrointestinal tract, but is absent from smooth muscle from several other tissue sources, skeletal muscle and cardiac muscle. The M2B3 glycoprotein is similar, but not identical, in apparent Mr to a transformation-associated glycoprotein, Q14. Further, the M2B3 and Q14 species are related antigenically. Nonetheless, M2B3 and Q14 are distinct glycoproteins based on clear differences in cell distribution and in partial peptide mapping. The M2B3 antigen described herein is sulfated on tyrosine, and represents one of the few cell surface proteins described to date that is sulfated on tyrosine residues. Our studies suggest the function of the M2B3 glycoprotein is likely to be associated with cell proliferation of cell adhesion.

HIV-1 infection and expression in human colonic cells: infection and expression in CD4+ and CD4- cell lines.

Three human colonic epithelial cell lines, SW620, HT29, and T84, were characterized with respect to HIV-1 infection and gene expression. SW620 and HT29, but not T84, could be infected with HIV-1. CD4 messenger RNA and its protein product were identified in SW620 cells but not in HT29 or T84 cells. Anti-CD4 antibody blocked infection of SW620 cells but had no effect on infection of HT29 cells. In SW620 and HT29 cells transfected with the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, an intact HIV-1 enhancer element was required for stimulation of CAT activity by tumor necrosis factor alpha (TNF alpha) and phorbol ester. T84 was not able to mediate a TNF alpha or phorbol ester response. These studies provide further evidence that HIV-1 can infect cells by mechanisms other than those mediated by the CD4 receptor and describe complementary models for analyzing HIV-1 infection and expression in colonic epithelial cells.

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland.

Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFalpha and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro-in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.