Human Fibroblast-conditioned Medium Research Articles

Nasal Fibroblast Conditioned Medium Promotes Cell Attachment and Migration of Human Respiratory Epithelium

Endoscopic sinus surgery (ESS) is a well-known surgical treatment for chronic rhinosinusitis disease after failed medical and antibiotics treatment. However, improper wound healing might induce synechiae or adhesion. Conditioned medium from cultured cells is known to promote wound healing and potentially able to accelerate wound healing in ESS and other airway epithelial injuries. This study was to investigate the effect of human nasal fibroblast conditioned medium on the attachment, proliferation and migration of respiratory epithelial cells (RECs) in an in vitro model. RECs and fibroblasts were co-cultured in Defined Keratinocytes Medium and F-12 and Dulbecco’s Modified Eagle’s Medium. Once confluent, the fibroblasts were removed, leaving the colonies of RECs to reach confluency. RECs and fibroblasts were cultured separately and the conditioned medium was acquired by culturing fibroblast either in DKSFM or F12: DMEM, denoted as NFCM_DKSFM and NFCM_FD, respectively. RECs were supplemented with 20% conditioned medium for attachment, proliferation and migration assay. The results showed significantly higher cell attachment in NFCM_DKSFM (3452.77±588.1 cell/cm2) compared to NFCM_FD (2336.1±440.4 cell/cm2) and DKSFM alone (2819.8±509.5 cell/cm2). After 7 days, the specific growth rate was higher in DKSFM (0.019±5.16×10-4 h-1) compared to NFCM_DKSFM (0.015±8.94×10-4 h-1) and NFCM_FD (0.013±1.03×10-3 h-1). The mean of migration rate was significantly higher in NFCM_DKSFM (4341.81±385.7 μm2/hr) compared to NFCM_FD (1803.38±408.1 μm2/hr) and DKSFM (1933.48±271.9 μm2/hr). Hence, NFCM_DKSFM supplementation provides suitable culture conditions for RECs through increased cell attachment and migration, which suggest that the factors secreted in conditioned medium may play a major role in enhancing airway epithelial wound healing.

Abstract 5083: Adipocytes promote pancreatic cancer cell proliferation and progression

Abstract Pancreatic cancer (PC) is a devastating disease with an extremely low survival rate. By the year 2030, this disease is projected to be the second deadliest cancer, bypassing breast, prostate and colorectal cancer-related fatalities. While population-based epidemiological studies have reported that obesity and a higher BMI (body mass index) not only increase the risk of developing PC but also promote tumor aggressiveness, and reduce overall patient survival, how obesity may influence pancreatic tumor progression and aggressiveness is not well understood. We hypothesize that the increased local presence of mature adipocytes (in obesity) within the pancreatic microenvironment markedly enhances the tumorigenic functions of pancreatic tumors and tumor-associated stroma, thereby accelerating tumor growth and progression. In this study, we used mature adipocytes and pancreatic cancer (PC) and pancreatic stellate (PSC) cell lines and a variety of in vitro functional assays including migration, proliferation, gemcitabine chemoresistance and development of a fibrotic phenotype, to determine whether local interactions between adipocytes and tumor cells promote PC growth and to identify the underlying mechanism/s. In cell migration assays, both PCs (MiaPaca-2, AsPC-1 and BxPC-3) and PSCs (HImPSC) migrated at significantly higher rates towards adipocyte conditioned medium (ACM) compared to serum-free or normal human fibroblast conditioned medium. The ACM-mediated stimulation of migration was dose-dependent and adipocyte-specific. ACM pretreatment of PC cells as well as PSCs resulted in a significant increase in proliferation rates. Given that the development of chemoresistance is a critical challenge in PC therapy, we next investigated the effects of adipocytes on PC cell gemcitabine toxicity. Pretreatment with ACM resulted in 50-70% increased cell viability for PC and PSC's in the presence of gemcitabine. Extensive fibrosis is a hallmark of pancreatic tumors and both PC and PSCs contribute to excessive ECM production in the PC microenvironment. Compared to cells treated with full medium, ACM-treated PSC and PC cells significantly upregulated their expression of fibronectin and Collagen VI. Taken together, our results indicate that secreted factor/s present in ACM promote cell migration, proliferation, gemcitabine resistance and induce a fibrotic phenotype in PC and PSC. Current efforts in our laboratory are focused on identifying the adipocyte-secreted factor/s that may mediate these effects. Our findings will establish a direct causal role for obesity in pancreatic cancer progression and identify molecular determinants that may drive this process, thereby delineating important pathways in the obesity-pancreatic cancer axis that may be therapeutically targeted in the future. [Supported by: The Advanced Medical Foundation] Citation Format: Roopali Roy, Marsha A. Moses. Adipocytes promote pancreatic cancer cell proliferation and progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5083. doi:10.1158/1538-7445.AM2015-5083

Expression of Local Immunosuppressive Factor, Indoleamine 2,3-dixygenase, in Human Coreal Cells

Purpose: To identify the localization of indoleamine 2,3-dioxygenase (IDO) in human corneal cells and to evaluate its ability to act as a local immunosuppressive factor. Methods: The expression profile of IDO was obtained with RT-PCR and Western blot of in a primary culture of human corneal cells (fibroblasts, epithelial cells and endothelial cells). In order to investigate the immunosuppressive function of IDO, immune cells were cultured in a human corneal cell-conditioned medium, and their prolifleration was identified by the MTT assay. Moreover, apoptotic effects of IDO in immune cells treated with IFN-γ were also investigated with apoptosis ELISA. Results: Among the three different types of human corneal cells analyzed, mRNA and protein expression of IDO was observed only in human corneal fibroblasts. Immune cells cultured in a human corneal fibroblast-conditioned medium showed inhibited proliferation. Moreover, IFN-γ-induced expression of IDO significantly enhanced apoptotic ability in a dose-depandant manner. Conclusions: Our results suggest that human corneal fibroblasts are relatively immuno-resistant and that expression of IDO may be one of the factors involved in the immune tolerance observed in corneal grafts.

Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination.

The Complement Component C1s Is the Protease That Accounts for Cleavage of Insulin-like Growth Factor-binding Protein-5 in Fibroblast Medium

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s purified from the conditioned medium cleaved C(4), a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.

Characterization of Cell-Associated and Soluble Forms of Connective Tissue Growth Factor (CTGF) Produced by Fibroblast Cells In Vitro

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate Mr 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-2601 peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non-or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to αα, αβ or ββ PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-α-R or -β-R, and was not antagonized by a neutralizing PDGF-R-α antiserum.These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

Cleavage Analysis of Insulin-like Growth Factor (IGF)-dependent IGF-binding Protein-4 Proteolysis and Expression of Protease-resistant IGF-binding Protein-4 Mutants

Cultured human fibroblasts and osteoblast-like cells secrete an insulin-like growth factor (IGF)-dependent protease that cleaves IGF-binding protein-4 (IGFBP-4) into two fragments of approximately 18 and 14 kDa. Edman degradation of the isolated proteins established the amino termini of the reaction products. Sequence analysis of the 14-kDa carboxyl-terminal half of IGFBP-4 suggested cleavage after methionine at position 135 of the mature protein. Four variant IGFBP-4 molecules with single amino acid substitutions around this cleavage site were constructed and expressed. Wild-type and mutant IG-FBPs-4 bound IGF-I and IGF-II with equivalent affinities and, in the intact state, were equally effective inhibitors of IGF-I action. However, the IGFBP-4 mutants were relatively resistant to IGF-dependent proteolysis. A 5-6-h incubation in human fibroblast conditioned medium in the presence of IGF-II was sufficient for near total hydrolysis of wild-type IGFBP-4, whereas the mutant IGFBPs-4 were only minimally affected at this time. After a 24-h incubation with IGF-II, all mutant IGFBPs-4 showed extensive proteolysis, generating 18- and 14-kDa fragments. Pre-exposure of human fibroblasts in serum-free conditioned medium to IGF-II for 5 h potentiated subsequent IGF-I stimulation of DNA synthesis. When added with IGF-II, the protease-resistant mutant IG-FBPs-4, but not wild-type IGFBP-4, suppressed IGF-II enhancement of IGF-I-stimulated DNA synthesis. These biological studies suggest that the IGFBP-4/IGFBP-4 protease system may play a role modulating local cellular response to IGF-I.

Development of motility of Langerhans cell through extracellular matrix by in vitro hapten contact.

To study the migration capacity of Langerhans cells in vitro, a reconstituted basement membrane invasion assay (Matrigel assay) was performed. Partially enriched human epidermal Langerhans cell suspensions (6-20%) were incubated with or without haptens [2,4,6-/trinitrobenzenesulfonic acid (TNBS) and fluorescein isothiocyanate (FITC)] or an irritant sodium lauryl sulfate (SLS) in Hanks' balanced salt solution for 10 min at 37 degrees C, and were then used for the invasion assay. Fibroblast-conditioned medium prepared from human dermal fibroblasts was used as a source of chemoattractants. Non-treated cells showed a gradual increase in migration of HLA-DR-positive cells through the Matrigel-coated filters in a time-dependent manner. In vitro TNBS and FITC treatment resulted in a significant stimulation of this migration. By contrast, SLS treatment did not increase the number of migrating cells. The migration capacity of hapten-treated cells decreased both in the absence of human fibroblast-conditioned medium and in the presence of this medium in the upper compartment of the chamber, indicating that this medium actually functioned as chemoattractant. Our findings suggest that the contact between hapten and Langerhans cells could be one of the triggers to initiate and/or stimulate Langerhans cell migration from the epidermis to the dermis. In addition, some factors derived from the dermal fibroblasts may promote and regulate the directional migration of Langerhans cells.

Phorbol ester tumor promoters regulate insulin-like growth factor-binding protein-4 proteolysis.

Cultured human fibroblasts secrete a specific protease that alters extracellular insulin-like growth factor-binding protein-4 (IGFBP-4) structure and function. This enzyme appears to be secreted in a latent form and requires IGFs for activation. To study regulation of the IGFBP-4 protease, we treated normal adult human fibroblasts with various hormones and growth regulatory factors, and collected the human fibroblast-conditioned medium (HFCM) for analysis of IGFBP-4 protease activity. The IGFBP-4 protease assay involved incubation of 50 microliters HFCM with or without 5 nM IGF-II for 6 h at 37 C under cell-free conditions; IGF-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting. In HFCM from cells treated with vehicle, GH, insulin, epidermal growth factor, steroid hormones, or forskolin, IGF-II induced the select loss of detectable IGFBP-4 during the assay. In contrast, IGFBP-4 levels were maintained when HFCM from cells treated with phorbol ester tumor promoters was incubated with IGF-II under cell-free conditions. Hydrolysis of [125I]IGFBP-4 to 18,000 and 14,000 mol wt fragments also was prevented in HFCM from cells treated with phorbol esters. Phorbol esters had no effect on endogenous or exogenous IGFBP-4 proteolysis when added directly to HFCM during the assay, however. Treatment of cells with actinomycin-D or cycloheximide could prevent a phorbol ester-induced block of IGF-dependent IGFBP-4 proteolysis. These data suggest that phorbol ester tumor promoters stimulate human fibroblasts to produce and secrete an inhibitor of the IGFBP-4 proteolytic reaction. Alterations in IGFBP-4 protease activity could affect local IGF action through regulation of IGFBP-4 availability.

Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.

Cultured human fibroblasts secrete insulin-like growth factor IA prohormone.

Nucleotide sequencing of the two known cDNAs encoding human insulin-like growth factor I (IGF-I) predicts the existence of two different prohormone forms of IGF-I. The E peptide regions extend the carboxy-terminus of IGF-I by either an additional 35 (IGF-IA) or 77 (IGF-IB) amino acids. With a specific and sensitive RIA employing an antiserum directed against a synthetic peptide that is unique to the E peptide region of IGF-IA prohormone (EIA), we have identified EIA-immunoreactive material in the conditioned medium of fetal and postnatal human fibroblasts in culture. Incubation of postnatal human fibroblasts with GH increased specific immunoreactive EIA secretion 2- to 3-fold. There was no immunologically detectable 7.5K IGF-I or IGF-II peptide in acid-chromatographed human fibroblast-conditioned medium under either basal or GH-stimulated conditions. Acid chromatography of human fibroblast-conditioned medium on Sephadex G-75 revealed a single elution peak of EIA immunoreactivity corresponding to a mol wt of 9-17 K. With neutral chromatography, EIA immunoreactivity eluted at 25-38K mol wt. These data suggest that the E peptide region of IGF-IA is translated and released as part of the prohormone form in cultured human fibroblasts, and that the levels of this prohormone are regulated by GH.

Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation.

Specific, high affinity insulin-like growth factor (IGF) binding proteins are secreted by human fibroblasts in culture. By multiple criteria, the species of IGF binding proteins produced by human fibroblasts are distinct from the HepG2/amniotic fluid IGF binding protein, but share many characteristics with the growth hormone-dependent IGF binding protein forms predominant in normal adult human plasma. Treatment of cultured human fibroblasts with growth hormone produced an increase in IGF binding protein activity in the medium, while addition of glucocorticoids markedly diminished IGF binding activity. Insulin, epidermal growth factor, platelet-derived growth factor, and progesterone had no effect on IGF binding activity in fibroblast media. In comparison, HepG2 IGF binding activity was enhanced by progesterone, decreased by insulin, and unaffected by growth hormone or glucocorticoid treatment. Five molecular forms of IGF binding proteins were identified by Western ligand blots in human fibroblast conditioned medium, with Mr = 41,500, 37,000, 32,000, 28,000, and 23,000. In human fibroblast conditioned medium, the Mr = 41,500 and 37,000 IGF binding protein species were abundant, as in normal human plasma, with a major Mr = 23,000 form which was a minor component in plasma.

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells.

Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role in angiogenesis and regeneration of vascular endothelium in vitro.

Human arterial wall cells secrete factors that are chemotactic for monocytes.

Macrophages and arterial smooth muscle cells comprise the cellular components of the atherosclerotic plaque. The vessel wall accumulation of macrophages occurs by a process of increased circulating monocyte migration into the vessel wall. In these studies it is demonstrated that human macrophages and arterial smooth muscle cells in culture secrete potent chemotactic factors for freshly isolated human monocytes. In contrast, human fibroblast-conditioned medium has no chemotactic activity. The effect of macrophage-conditioned medium is a function of macrophage differentiation and can be potentiated by macrophage activation. These results suggest that secretory products of human macrophages and arterial smooth muscle cells may be important stimuli for increased monocyte migration into the vessel wall in vivo.

Human fibroblast-conditioned medium contains a 100K dalton glucose-regulated cell surface protein.

Abstract This report describes the identification and partial characterization of a 100K dalton cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (conditioned medium) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.