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Early studies have indicated that estrogen receptor beta (ERβ) can influence the progression of clear cell renal cell carcinoma (ccRCC). Here, we report the mechanistic details of ERβ-mediated progression of ccRCC. ERβ increased ccRCC cell invasion via suppression of circular RNA ATP2B1 (circATP2B1) expression by binding directly to the 5' promoter region of its host gene ATPase plasma membrane Ca2 + transporting 1 (ATP2B1). ERβ-suppressed circATP2B1 then led to reduced miR-204-3p, which increased fibronectin 1 (FN1) expression and enhanced ccRCC cell invasion. Targeting ERβ with shRNA suppressed ccRCC metastasis in a murine model of RCC; adding circATP2B1 shRNA partly reversed this effect. Consistent with these experimental results, ccRCC patient survival data from The Cancer Genome Atlas indicated that a patient with higher ERβ and FN1 expression had worse overall survival and a patient with higher miR-204-3p expression had significantly better overall survival. Together, these results suggest that ERβ promotes ccRCC cell invasion by altering the ERβ/circATP2B1/miR-204-3p/FN1 axis and that therapeutic targeting of this newly identified pathway may better prevent ccRCC progression. Significance: These results identify an ERβ/circATP2B1/miR-204-3p/FN1 signaling axis in RCC, suggesting ERβ and circular RNA ATP2B1 as prognostic biomarkers for this disease. Cancer Res; 78(10); 2550-63. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5β1 integrin-dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5β1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment., (©2012 AACR.)

The switch of tumor cells from an epithelial to a mesenchymal-like phenotype [designated as epithelial-to-mesenchymal transition (EMT)] is known to induce tumor cell motility and invasiveness, therefore promoting metastasis of solid carcinomas. Although multiple studies have focused on elucidating the signaling events that initiate this phenotypic switch, there has been so far no characterization of the pattern of soluble mediators released by tumor cells undergoing EMT, and the potential impact that this phenotypic switch could have on the remodeling of the tumor microenvironment. Here we show that induction of EMT in human carcinoma cells via overexpression of the transcription factor Brachyury is associated with enhanced secretion of multiple cytokines, chemokines, and angiogenic factors and, in particular, with the induction of the IL-8/IL-8R axis. Our results also indicate the essential role of interleukin 8 (IL-8) signaling for the acquisition and/or maintenance of the mesenchymal and invasive features of Brachyury-overexpressing tumor cells and show that IL-8 secreted by tumor cells undergoing EMT could potentiate tumor progression by inducing adjacent epithelial tumor cells into EMT. Altogether, our results emphasize the potential role of EMT in the modulation of the tumor microenvironment via secretion of multiple soluble mediators and suggest that IL-8 signaling blockade may provide a means of targeting mesenchymal-like, invasive tumor cells.

Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha5beta1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha5beta1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha5beta1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha5beta1-integrin as targets for breast cancer therapy., (Copyright 2010 AACR.)

Aberrant microenvironments and loss of balance in cell-extracellular matrix signaling are associated with breast cancer invasion, metastasis, and resistance to therapy. We have recently shown that increased beta1 integrin signaling is involved in malignant progression and that inhibitory antibody to beta1 integrin leads to selective apoptosis and decreased proliferation in three-dimensional cultures and in xenograft models of breast cancer in vivo. To investigate the clinical importance of these findings, in the present study we examined the expression of beta1 integrin and extracellular beta1 integrin ligands fibronectin and laminin-1 in a cohort of 249 breast cancer patients who had a median follow-up of 8.4 years. Among the 149 scorable cases, the highest beta1 integrin intensity score (3+ versus 0-2+) was associated with significantly decreased 10-year overall survival of 48% versus 71% (P<0.03) and decreased disease-free survival of 50% versus 80% (P<0.05). Importantly, high fibronectin expression was associated with decreased overall and disease-free survival on univariate analysis (P<0.04) and beta1 integrin intensity score was significantly correlated with fibronectin expression (Kendall's tau-b=0.19; P=0.03). In a multivariate Cox proportional hazards model, beta1 integrin intensity score remained a significant independent predictor of overall survival [hazard ratio (HR), 1.69; 95% confidence interval (95% CI), 1.19-2.38; P<0.003] and disease-free survival (HR, 1.87; 95% CI, 1.21-2.88; P<0.005). These findings show that beta1 integrin expression has potential prognostic value in invasive breast cancer and that coexpression of fibronectin may help identify patients with more aggressive tumors who may benefit from targeted therapy.

Dipeptidyl peptidase IV (DPPIV) is a serine protease with tumor suppressor function. It regulates the activities of mitogenic peptides implied in cancer development. Progression of benign prostate cancer to malignant metastasis is linked to increased production of basic fibroblast growth factor (bFGF), a powerful mitogen. In this study, using in vitro model system we show that DPPIV loss is associated with increased bFGF production in metastatic prostate cancer cells. DPPIV reexpression in prostate cancer cells blocks nuclear localization of bFGF, reduces bFGF levels, inhibits mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)1/2 activation, and decreases levels of urokinase-type plasminogen activator, known downstream effectors of bFGF signaling pathway. These molecular changes were accompanied by induction of apoptosis, cell cycle arrest, inhibition of in vitro cell migration, and invasion. Silencing of DPPIV by small interfering RNA resulted in increased bFGF levels and restoration of mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK)1/2 activation. These results indicate that DPPIV inhibits the malignant phenotype of prostate cancer cells by blocking bFGF signaling pathway.

Previous results using gene transfection methods have shown that the wild-type (WT) von Hippel-Lindau (VHL) gene can function as a potent tumor suppressor gene in vivo for renal cell carcinoma (RCC) cells in the absence of any suppressive effect on cell growth in monolayer cell culture under serum-rich conditions. Because we had previously found that the function of some oncogenes, such as mutant ras, can be influenced by three-dimensional growth as multicellular spheroids (J. Rak et aL, J. Cell Biol., 131: 1587-1598, 1995), we reasoned the same might be true for suppressor genes as well. We, therefore, decided to compare and study the effects of the WT VHL gene in monolayer versus three-dimensional culture systems of the RCC cell line 786-0, which contains an inactivated VHL gene. We found that the reintroduction of the WT VHL gene into mutant VHL RCC cells resulted in growth suppression in vitro, but only when the cells were grown as spheroid cultures. This decrease in cell proliferation was associated with several features of cell differentiation/morphogenesis, as shown by light and electron microscopy. Thus, in contrast to cultures of mutant VHL RCC cells, which formed very compact and cohesive spheroids, the WT VHL transfectants were loosely arranged and formed a network of tubular and trabecular structures within the spheroids. The morphological changes of the WT VHL spheroids were associated with the deposition of fibronectin in the extracellular space, a feature that was absent in the mutant and inactivated VHL gene-expressing spheroids. The results suggest the VHL gene may be involved in the maintenance of the epithelial phenotype of renal tubular cells, ie., it may act as a differentiation/morphogenetic factor. Moreover, this effect in tumors cells appears to be highly dependent on multicellular growth conditions that mimic the basic nature of solid tumors, such as RCC.

Loss of fibronectin (FN) from the cell surface has been shown to be closely associated with malignant transformation of cells. To elucidate the role of the FN matrix in the modulation of malignant phenotypes, we overexpressed a full-length cDNA encoding plasma-type FN in HT1080 human fibrosarcoma cells. The cells overexpressing FN adopted a more flattened morphology and deposited a moderately developed FN matrix both in vitro and in vivo, although the level of expression of integrin alpha5beta1 remained unchanged. FN-overexpressing cells exhibited a reduced cell motility on the substratum and grew poorly when injected s.c. into nude mice. Overexpression of FN also suppressed the ability of the tumor cells to proliferate in soft agar, whereas the suppression was reversed by inclusion in soft agar of the Arg-Gly-Asp (RGD)-containing peptide and adhesion-blocking antibodies against the central cell-binding domain of FN. Neither cell motility nor growth potential was altered by overexpression of a truncated form of FN lacking the central cell-binding domain. These results, taken together, indicate that increased deposition of FN in the pericellular matrix per se can suppress the motility and growth potential of tumor cells through interaction with RGD-recognizing integrins, most likely alpha5beta1.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

A serotonin-secreting human pancreatic carcinoid cell line (BON) is demonstrated to express transcripts for all three mammalian types of transforming growth factor beta (TGF beta 1, 2, and 3). Similarly, freshly excised carcinoid tumors from six patients were also found to express mRNA for all three of the type-beta TGFs. Medium conditioned by BON cells had detectable TGF beta activity, although most of the activity was latent as determined by radioreceptor assay with and without prior acid treatment. However, nonactivated BON-conditioned medium stimulated DNA synthesis, soft agar growth, and an increase in TGF beta 1 and fibronectin mRNA expression in AKR-2B fibroblasts. In addition, BON-conditioned medium had a potent endothelial cell growth-stimulatory activity. Since the TGF beta s inhibit growth of endothelial cells, the presence of other growth factors was suspected. TGF alpha, c-sis, and basic fibroblast growth factor transcripts were also found to be expressed by the BON carcinoid cells. These data indicate that multiple peptide growth factors may have a paracrine role in the desmoplastic reaction accompanying carcinoid tumors.

Clonal cell lines were derived from rat liver epithelial cells following their transformation with either v-raf or v-raf/v-myc. Cells transformed with v-raf alone showed reduced tumor incidence and tumor growth rates when implanted into nude mice, compared to cells also expressing the v-myc oncogene. A series of additional clones isolated from a tumor obtained following inoculation of an athymic nude mouse with the v-raf-transformed rat liver epithelial cells displayed an intermediate range of tumor aggressiveness. These findings indicate that unknown genotypic and/or phenotypic changes occur during tumor formation in vivo, which are required in addition to raf activation for complete expression of the malignant phenotype. This in vitro model of tumor progression was used to examine alterations in the expression of genes related to the growth control of liver epithelial cells, which may be involved in the malignant conversion of the preneoplastic cells. A close association was observed between the increased level of expression of the transforming growth factors alpha and beta 1, the decreased expression of extracellular matrix proteins fibronectin and collagen I, and the tumor aggressiveness (latency/growth rate), suggesting a causal role for these factors in the progression of v-raf-transformed rat liver epithelial cells to the fully malignant phenotype.

Alternative splicing of fibronectin pre-mRNA at the ED-A region has been shown to be deregulated in malignant human liver tumors (F. Oyama et al., J. Biol. Chem., 264: 10331-10334, 1989). In order to extend this observation to other human cancers, we investigated the splicing patterns of fibronectin pre-mRNA at both ED-A and ED-B regions in normal, fetal, and cancerous lung tissues. Unlike in the liver, the ED-A+ mRNA was constitutively expressed in the lung irrespective of ontogenic or oncogenic stages. Although fetal tissues expressed the ED-A+ mRNA slightly more than did adult tissues, there was virtually no significant difference between malignant and nonmalignant tissues in the level of the ED-A+ mRNA. In contrast, significant expression of the ED-B+ mRNA was observed with fetal and cancerous tissues but not with normal adult tissues. Increased expression of the ED-B+ mRNA was associated with all types of lung cancer including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. These results indicate that it is the ED-B, but not the ED-A, region where the alternative splicing of fibronectin pre-mRNA is oncodevelopmentally regulated in the lung. Our results also suggest that deregulation of the tissue-specific alternative splicing of fibronectin pre-mRNA is not a unique phenotype of liver cancer but rather a general feature of naturally occurring human cancer.

Recent studies of murine tumor models and certain human tumor cell lines have provided evidence for intratumor heterogeneity in expression of extracellular matrix receptors and in the elaboration of matrix-degrading enzymes. However, little is known about possible intratumoral heterogeneity in the production of matrix macromolecules. We have, therefore, examined the biosynthesis and secretion of matrix proteins by cells derived from a polyclonal human cell line (JH-17) established from a large cell undifferentiated carcinoma of the lung. For the present studies, we focused on the production of collagens and structural glycoproteins by two phenotypically different aneuploid clones, designated C13 and C22. These clones were distinctive in their inability to grow in soft agar or to form tumors in nude mice and had identical DNA contents. Tumor cells were labeled with [3H]proline and the newly synthesized proteins accumulating in the culture medium were identified using biochemical and immunologic techniques. Clone C13 secreted at least three genetically distinct collagens, including type V procollagen (PC), type IV procollagen, and a type VIII-like collagen. By contrast, the clone C22 synthesized fibronectin, and a single bacterial collagenase-sensitive and pepsin-resistant component consistent with type I trimer. These studies emphasize the potential diversity of matrix proteins synthesized by neoplastic cells and suggest that there is intratumoral heterogeneity in matrix protein biosynthesis in vivo. These studies further suggest that tumor-derived matrix may be altered during tumor progression or cell selection in vivo.

Laminin, a glycoprotein antigenically similar or identical to a component of epithelial basement membranes, was identified as a major component of the abundant extracellular matrix synthesized by an experimentally induced rat yolk sac tumor. Immunocytochemical staining revealed laminin in cultured tumor cells as well as in their extracellular matrix. The presence of soluble laminin in the culture media of the tumor cells was demonstrated using metabolic labeling followed by identification by immunoprecipitation and sodium dodecyl sulfate:polyacrylamide gel electrophoresis. This revealed two polypeptides with molecular weights of approximately 200,000 and 400,000. These comigrated with the polypeptides of mouse laminin isolated previously. The yolk sac tumor tissue grown in vivo contained laminin in the tumor cells and in the extracellular material as evidenced by immunofluorescence and immunoperoxidase staining. Immunization with the tumor matrix resulted in an antiserum that contained antilaminin and natifibronectin and was made specific for laminin by absorption with fibronectin. This antiserum precipitated laminin polypeptides from culture medium of yolk sac tumour cells and stained basement membranes in rat tissues in a manner indistinguishable from antilaminin. The presence of laminin in rat yolk sac cells, the presumed origin of our yolk sac tumor, was studied in some detail. Laminin was found to be present in normal cells of the visceral as well as the parietal yolk sac layer and in their basement membranes suggesting, but not proving, that both types of cells have ability to synthesize laminin. Production of laminin and the presence of laminin-containing basement membrane material may be important for the biological behavior of the yolk sac tumor. This tumor will also be a useful source of laminin for chemical and biological characterization of this basement membrane protein.

Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-[35S]methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. We had shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

Human ecto- and endocervical uterine epithelia grow in culture as stratifying and nonstratifying cell layers, patterns reflecting the differentiation of the corresponding cells in vivo. We found that both cultures synthesized and secreted fibronectin but very little other known glycoproteins of basal laminae, laminin, and type IV collagen. Fibronectin production was observed only in the lining marginal cells of the expanding periphery of ectocervical islands, but differentiating and keratinizing cells had loss fibronectin. Most cells in endocervical epithelial cultures grew as a monolayer and produced fibronectin. After stratification of the ectocervical cells, the cytokeratin polypeptide pattern switched to the large-molecular-weight type. In endocervical cultures, the low-molecular-weight keratin polypeptides persisted. Fibronectin production thus appears to be linked to the state of differentiation of the epithelial cells, and fibronectin may function in the attachment of the basal cells to underlying basal lamina matrix.

The ability of the human germ cell tumor cell lines Tera 1, Tera 2, PA-1, LICR LON HT 39/7, LICR LON HT3B1, and LICR LON HT 53 to synthesize and secrete fibronectin has been studied. The presence of cellular fibronectin was examined using indirect immunofluorescence, whereas the synthesis and secretion of the protein were studied using specific immunoprecipitation from cultures radioactively labeled with [35S]methionine. Two of the cell lines, LICR LON HT 39/7 and Tera 1, did not synthesize fibronectin, whereas all the other cell lines did. Plasma membrane fibronectin could not be demonstrated on any of the cell lines, although cytoplasmic fibronectin was easily demonstrable. The cells appear therefore to synthesize fibronectin but not retain it. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the secreted fibronectin produced by the human teratoma cell lines showed that it had an apparent molecular weight greater than that produced by adult human breast fibroblasts or human plasma fibronectin. Peptide mapping of this secreted germ cell tumor fibronectin, by partial proteolytic cleavage, yielded peptide patterns similar to those obtained from either human plasma fibronectin or adult human breast fibroblast fibronectin. The difference in molecular weight between the fibronectins may therefore be due to changes in their patterns of glycosylation.

Human epithelial cell lines derived from both carcinomatous and nomalignant tissues were characterized with respect to the presence and distribution of fibronectin by immunofluorescence microscopy. In cell lines derived from nonmalignant tissues or from primary carcinomas, fibronectin was found predominantly in an extracellular matrix. In contrast, cell lines derived from metastatic carcinomas displayed very little or no fibronectin. Metabolic labeling studies indicated that a positive line synthesized fibronectin de novo rather than absorbing the protein from the media. Negative lines neither synthesized fibronectin nor secreted it into the culture fluid, suggesting that they were not producing fibronectin. Evidence is presented that cells in culture change their properties after extensive subculture since a small amount of fibronectin in an extracellular matrix was observed after extensive subculture of two metastatic lines that were originally negative.

The cell surfaces of human colon cancer cells before and after exposure to N,N-dimethylformamide (DMF) were probed using radioiodination and immunofluorescent labeling techniques. Growth of the human colon carcinoma cell line HCT MOSER in DMF-supplemented culture medium resulted in monolayer culture growth and marked cell morphology alterations consisting of cellular flattening and elongation. Accompanying the morphology alterations were distinct changes in the cell surface protein composition as determined by 125I labeling and electrophoresis. The cell surface changes associated with growth of HCT MOSER cells in the presence of DMF were dependent upon time of exposure to DMF and DMF concentration. Furthermore, removal of DMF-treated HCT MOSER cells from DMF-containing growth medium caused reversion of both cell morphology and cell surface composition to a state comparable to that of cells not exposed to DMF. The HCT MOSER cell surface alterations produced by DMF included a reduction of radioiodinated surface proteins with molecular weights of 87,000, 120,000, and 180,000 and an increase of a 125I-labeled surface protein with a molecular weight of 200,000-250,000. Appearance of a surface protein of approximately 200,000 molecular weight and assumption of a fibroblast-like morphology by DMF-treated HCT MOSER cells suggested that this approximately 200,000 molecular weight material might be fibronectin. Immunofluorescent labeling with anti-human fibronectin showed that HCT MOSER cells grown in DMF did manifest an anti-fibronectin immunoreactive material that was only transiently associated with the cell surface before being released. DMF-treated HCT MOSER cultures continued to express surface carcinoembryonic antigen, indicating that the presence of material immunoreactive with anti-human fibronectin was not secondary to proliferation of a contaminating fibroblast population. The response of HCT MOSER cells to DMF paralleled in many ways that previously reported for methylcholanthrene-transformed AKR-2B (AKR-MCA) fibroblasts. However, unlike AKR-MCA cells, HCT MOSER cells did not exhibit an increase in 125I incorporation per microgram DNA as a function of time of exposure to DMF, which suggests that the surface protein with a molecular weight of approximately 200,000 induced by DMF was not retained on the cell surface.