Inhibition of transforming growth factor alpha stimulation of human squamous cell carcinoma of the head and neck with anti-TGF-alpha antibodies and tyrphostin.

BACKGROUND: Transforming growth factor alpha (TGF-alpha) and its receptor (EGF-R) may regulate normal and malignant epithelial cell growth by an autocrine mechanism. We investigated the role of TGF-alpha in regulating head and neck SCC tumor growth. METHODS: TGF-alpha and EGF-R levels were measured in 7 SCC cell lines and 14 SCC biopsies by RIA, Scatchard, and Western analysis. TGF-alpha autocrine stimulation of DNA synthesis in SCC cell lines was assessed by incubation with TGF-alpha neutralizing antibodies and tyrphostin AG 1478, a selective and potent inhibitor of EGF-R kinase. RESULTS: All SCC cell lines synthesized TGF-alpha and expressed elevated EGF-R levels compared to normal keratinocytes. Twelve of the 14 SCC biopsies contained TGF-alpha protein and 8 had specific EGF-R. Exogenous TGF-alpha or EGF significantly increased DNA synthesis in 4 of 5 SCC cell lines. TGF-alpha neutralizing antibodies or tyrphostin AG 1478 reduced DNA synthesis in the two SCC cell lines (FaDu and SCC9) tested. CONCLUSIONS: These results indicate that SCC cell lines and tumors usually synthesize TGF-alpha, have elevated levels of EGF-R, and are mitogenically stimulated by a TGF-alpha autocrine system. Selective inhibition of the TGF-alpha system by EGF-R kinase inhibitors or TGF-alpha neutralizing antibodies may be useful strategies for treating SCC that overexpress TGF-alpha and its receptor.

Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats.

Epithelial cell lines from the proximal tubule of SHR and WKY rats were generated by microdissection, cell growth on 3T3 cell feeder layers, and transduction of the SV40 large T-antigen gene. The cell lines that formed confluent, electrically-resistive monolayers (basal conductance 1 to 20 mS/cm2) were selected for further study. Of these, cell lines generated from one rat did not show evidence of T-antigen expression or integration, and apparently immortalized spontaneously. Cell lines from three other rats expressed high levels of T-antigen, and showed evidence of integration of one or more copies of T-antigen. All cell lines formed polarized monolayers with apical microvilli, tight junctional complexes, and convolutions of the basolateral plasma membrane. Most cell lines grew in the absence of extracellular glucose indicating a capacity for gluconeogenesis. Sodium succinate cotransport and P2-purinergic receptor mediated signaling were demonstrated in all lines tested. The cell lines also showed that Na/H exchanger activity is regulated by angiotensin II. The results indicate that these cell lines express a proximal tubular phenotype, and are morphologically and functionally similar to primary cultures. These rat cell lines represent a new, potentially useful cell model for elucidating the cellular and molecular mechanisms of genetic differences in proximal tubule Na+ reabsorption.

New methods for maintaining human renal epithelial cells and analyzing their ion transport functions: potential analysis of genetic disease.

OBJECTIVES: New methods are available to immortalize parenchymal cells from exocrine glands and kidney with retention of differentiation. Adaptation of this technology to small, single-donor biopsy material or surgical specimens could provide genetically homogeneous cells for functional analyses and correlation with genetic background and underlying biochemistry. To develop a methodology useful for renal sodium metabolism, epithelial cell line generation was tested in a hypertensive rat model with features similar to salt-sensitive hypertension in humans. This form of hypertension has a large genetic component and is prevalent in African Americans. DESIGN: Protocols were designed to immortalize primary cultures of microdissected proximal tubule epithelial cells from spontaneously hypertensive (SHR) and control, normotensive Wistar-Kyoto (WKY) rats. Immortalization was based on a replication-defective retrovirus coding for SV40 large T-antigen as positive cell cycle regulator. Transport competent cells that grow on porous filters to form confluent monolayers were selected. RESULTS: Several proximal tubule cell lines have been developed from SHR and WKY rats. The cells retain important differentiated features, such as epithelial polarity, low monolayer conductance, and sodium-succinate cotransport. They are suitable for analyses of electrolyte transport by electrophysiology or imaging of intracellular fluorescent indicator dyes, such as sodium-binding benzofuran isophthalate. CONCLUSION: Feasibility of generating epithelial cell lines from defined renal segments was demonstrated. The cells retain important transport function so that analyses of sodium metabolism and the influence of genetic background on it are possible. The methodology is applicable to human specimens.

Detection of epidermal growth factor and transforming growth factor alpha protein in meningiomas and other tumors of the central nervous system in human beings.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens for normal cells of ectodermal and mesodermal origin. Evidence is accumulating that suggests that EGF, TGF alpha and their common receptor (EGF/TGF alpha-R) influence development and functioning of tissues of the central nervous system (CNS). To further investigate the possible roles of EGF, TGF alpha and their receptor in autocrine/paracrine regulation of tumor growth in the CNS, a series of tumors of the CNS were analyzed for the presence of specific, high affinity EGF/TGF alpha receptors and for the presence of immunoreactive TGF alpha protein. Binding of 125I-EGF to crude membranes from a pool of meningiomas was competed for equally well by low concentrations of unlabeled EGF or TGF alpha, but not by high concentrations of other protein hormones, demonstrating the high degree of specificity of the EGF/TGF alpha receptor. Specific binding of 125I-EGF was dependent upon time and temperature, with maximum specific binding achieved after two hours at 22 degrees C. Scatchard analysis of six tumors of the CNS large enough to permit titration analysis generated linear plots with an average kilodalton of 1.1 +/- 0.1 nanometer (+/- standard error of the mean), suggesting the presence of a single class of EGF/TGF alpha-R with high affinity. EGF also stimulated phosphorylation of a 170 kilodalton protein in membrane fraction of a meningioma, demonstrating that the EGF/TGF alpha-R in this tumor retained EGF-stimulated kinase autophosphorylating activity. Membranes for 17 additional smaller tumors of the CNS were analyzed for specific binding of 125I-EGF by single, high concentration method, and all 17 tumors were found to contain specific binding of 125I-EGF. The average level of 125I-EGF for all 23 tumors of the CNS was 46 +/- 27 femtomoles per milligram protein with a range of 1 femtomoles per milligram for both a pituitary adenoma and meningioma to 638 femtomoles per milligram for a glioblastoma. A series of 13 tumors of the CNS were analyzed for EGF alpha with use of a specific radioimmunoassay. TGF alpha immunoreactive protein was detected in all four malignant tumors of the CNS assayed at an average level of 2.6 +/- 1.1 nanograms per milligram soluble protein, whereas TGF alpha immunoreactive protein was detected in only two of nine benign tumors of the CNS. These results add support to the hypothesis that TGF alpha and its receptor may act by autocrine/paracrine mechanisms to influence growth of tumors of the CNS in vivo.

Correlation of hyaluronic acid accumulation and the growth of preneoplastic mammary cells in collagen: a longitudinal study.

Hyaluronic acid accumulation is characteristic of mammary tumor cells, and the amount that accumulates seems to correlate with the degree of malignancy of the producing cells. We have tested directly the relationship between hyaluronic acid accumulation and the replication rate of preneoplastic mammary cells in culture. We used nontumorigenic but immortal CL-S1 mouse mammary cells that were derived from a hyperplastic alveolar nodule. Using a collagen gel culture system, we found clear differences in the growth properties of cells before and after Passages 68 to 70. Late passage cells replicated earlier and faster than early passage cells in collagen and on plastic. The rate of cycling resembled that of tumorigenic mouse mammary cells during the first week of culture. Cells seeded at low densities cycled faster than those seeded at high densities during the second week in culture. Exogenous hyaluronic acid, at 10 to 1000 micrograms/ml, neither enhanced nor inhibited CL-S1 cell growth significantly in collagen, regardless of passage. However, by the third day in collagen, late passage cells produced 7 times more total glycosaminoglycans and 12 times more hyaluronic acid per cell than did early passage cells. Late passage cells also deposited 12 times more labeled hyaluronic acid in the matrix than did early passage cells, on a per-cell basis. After a decline in the deposition of hyaluronic acid in the extracellular matrix, growth ceased. The late passage cells did not grow in soft agar, indicating that they had not become neoplastic spontaneously during passage. However, their accelerated growth rate, coupled with the synthesis and secretion of large amounts of hyaluronic acid into the extracellular matrix, may characterize a distinct step in tumor progression in preneoplastic CL-S1 cells.

Growth factors and corneal endothelial cells: I. Stimulation of bovine corneal endothelial cell DNA synthesis by defined growth factors.

Peptide growth factors and other physiological growth modifiers were evaluated for their ability to stimulate DNA synthesis in early passage cultures of bovine corneal endothelial cells (BCEC). Increasing concentrations of newborn bovine serum (0.5-10%) causes a progressive increase in DNA synthesis, which approached a plateau at 10% serum. Supplementing medium with 10% serum from different lots of newborn bovine serum or fetal bovine serum stimulated significantly different levels of DNA synthesis by BCEC. Addition of epidermal growth factor (EGF) (2 nM) to medium containing 10% newborn or fetal bovine serum further increased DNA synthesis. Dose-response curves for EGF, transforming growth factor-alpha, basic fibroblast growth factor (bFGF), and insulin-like growth factor I showed that each significantly stimulated high levels of DNA synthesis (200-700% increase) compared with BCEC cultured in serum-free medium. Vaccinia growth factor, insulin, and transforming growth factor-beta each significantly stimulated lower levels of DNA synthesis (30-200% increase), whereas nerve growth factor, multiplication stimulating activity, and platelet-derived growth factor all failed to significantly stimulate DNA synthesis above the level of serum-free medium. Other physiological growth modifiers were tested for their effects on DNA synthesis of BCEC. Transferrin and low levels of 3',5'-cyclic monophosphate (cAMP) stimulated very low levels of DNA synthesis (50% increase) whereas linoleic acid, high levels of selenium, or cAMP each inhibited DNA synthesis 25-75% below the level of BCEC cultured in serum-free medium. A series of eight formulations containing various combinations of EGF, FGF, insulin, transferrin, selenium, linoleic acid, retinoic acid, cAMP, heparin, and endothelial cell growth factor were tested for their mitogenic action on BCEC cultures. A formulation containing EGF, insulin, transferrin, selenium, and linoleic acid (EGF + ITSL) stimulated the highest level of DNA synthesis of BCEC, which was approximately 25% higher than the increase stimulated by addition of 10% newborn bovine serum. The formulation consisting of EGF + ITSL was also evaluated as a supplement to corneal storage media. Addition of EGF + ITSL to three corneal storage media (McCarey-Kaufman, K-Sol, CSM) significantly stimulated increases in cell numbers of approximately 50% above the unsupplemented corneal storage media. These results demonstrate that BCEC respond selectively to different defined peptide growth factors and physiological growth modifiers, and suggest that supplementation of corneal storage media with a defined formulation (EGF + ITSL) may enhance corneal endothelial cell density.

Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells.

Epidermal growth factor (EGF) is a potent mitogen for corneal endothelial cells and may play a role in endothelial wound healing. To further characterize the interaction of EGF with endothelial cells, we measured biochemical parameters of 125I-EGF binding to cultured bovine corneal endothelial cells (BCEC), determined the pattern of EGF-induced protein phosphorylation, and investigated the influence of retinoic acid (RA) and transforming growth factor beta (TGF-beta) on EGF-induced DNA synthesis and receptor levels. Binding of 125I-EGF to BCEC was dependent on time, reaching a plateau after approximately 2 h at 37 degrees C, was specific for EGF, and had high affinity (Kd = 100 pM) with approximately 21,000 receptors per cell. Cellular substrates for the EGF receptor kinase, which may function as initial second messengers for EGF, were detected by autoradiography of sodium dodecyl sulfate polyacrylamide gels of 32P-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM, 100 nM, and 10 microM RA to BCEC cultured in serum-free medium for 24 h progressively inhibited DNA synthesis by up to 80% compared with control cultures. However, when added in combination with 5 nM EGF, 1 nM and 100 nM RA synergistically stimulated DNA synthesis by up to 80% above the level of EGF stimulation without altering EGF receptor levels or binding affinity. Thus, short-term exposure of BCEC to RA potentiated EGF-stimulated DNA synthesis, most likely by acting at a postreceptor step.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth factors and corneal endothelial cells: III. Stimulation of adult human corneal endothelial cell mitosis in vitro by defined mitogenic agents.

Human corneal endothelial cells (HCEC) do not mitose extensively in vivo after damage to the endothelial layer. However, HCEC will divide in vitro if cultured under appropriate conditions. We measured the ability of various sera, plasma, growth factors, and nutritional substances to stimulate mitosis of HCEC during 5 days of organ culture after a central freeze injury to the endothelium. Supplementation of a chemically defined medium (CDM) with 20% fetal human serum (FHS) induced significantly higher numbers of mitotic figures or labeled nuclei of human or cat corneas compared with paired corneas cultured in CDM alone. Furthermore, addition of 20% FHS produced more labeled nuclei than did addition of 20% fetal bovine serum or 20% adult human serum. Dialyzed fetal human serum failed to stimulate mitosis, indicating that one or more components of fetal human serum with molecular weight less than 12,000 are essential for mitosis. Human plasma also failed to stimulate mitosis, but an extract of human platelets significantly stimulated high levels of nuclear labeling, suggesting that growth factors contained in platelet granules were responsible for serum-stimulated mitosis of HCEC. Addition of 100 nM epidermal growth factor (EGF) or 10 microM insulin to CDM supplemented with low levels of adult human serum (0.5%) stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured with 0.5% adult human serum. Supplementation of corneal storage media (K-Sol and CSM) with a mixture of chemically defined agents consisting of EGF, insulin, transferrin, selenium, linoleic acid, and albumin stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured in the unsupplemented corneal storage media.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of biological activity of synthetic fragments of transforming growth factor-alpha.

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.

The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta.

Highly purified lysosomes, rough and smooth endoplasmic reticulum, and Golgi apparatus, as well as microvillus plasma membranes, bound 125I-labelled epidermal growth factor ([125I]EGF) with similar affinity. Scatchard plots for all the organelles were curvilinear. The apparent number of available binding sites per mg protein of intracellular organelles was 27-71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded by any of the organelles. Binding and dissociation of [125I]EGF in all organelles were dependent on the time and temperature of incubation. The specificity of [125I]EGF binding was similar in all organelles. The optimal pH for binding to lysosomes was 6.0, in contrast to 7.0 for all the other organelles. Exposure of different organelles to enzymes and protein-modifying reagents resulted in numerous binding differences between the intracellular organelles and microvillus plasma membranes. Covalent affinity labelling with [125I]EGF revealed two major proteins of 155 and 140(X10(3)) Mr in all the organelles. The 155 X 10(3) Mr protein was labelled predominantly in all organelles except rough endoplasmic reticulum, where both proteins were equally labelled. Addition of proteolytic inhibitors during isolation of organelles did not alter the pattern of [125I]EGF-labelled binding proteins found in the organelles. EGF also stimulated phosphorylation of the 155 and 140(X10(3)) Mr proteins in all the organelles. The 155 X 10(3) Mr protein was phosphorylated more than the 140 X 10(3) Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140 X 10(3) Mr protein was phosphorylated more than the 155 X 10(3) Mr protein in lysosomes and both proteins were equally phosphorylated in rough endoplasmic reticulum. Several organelles also contained minor [125I]EGF-binding proteins that did not show phosphorylation response and proteins that showed phosphorylation response but did not bind [125I]EGF. Thus, the present study demonstrates by a number of different criteria, that several intracellular organelles of term human placenta also contain EGF-binding and kinase activities.

Topography of human placental receptors for epidermal growth factor.

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

Effect of growth factors with dexamethasone on healing of rabbit corneal stromal incisions.

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal growth factor (EGF, 0.5 mg ml-1), fibroblast growth factor (FGF, 20 micrograms ml-1), and insulin (0.5 mg ml-1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml-1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54 +/- 4 g mm-1) or treated with EGF and dexamethasone (32 +/- 9 g mm-1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3 +/- 1 g mm-1) or dexamethasone (1 +/ 0 g mm-1) (P less than 0.001). The combination of dexamethasone with EGF significantly (P less than 0.025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28 +/- 8 g mm-1) or in combination with dexamethasone (25 +/- 7 g mm-1) was significantly increased compared with saline- or dexamethasone-treated corneas (P less than 0.001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin (P less than 0.01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5 +/- 4 g mm-1) or in combination with dexamethasone (2 +/- 1 g mm-1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [3H]-thymidine incorporation into pure cultures of human corneal fibroblasts (HCF) in defined culture medium. EGF (5 mM) or FGF (100 ng ml-1) alone stimulated [3H]-thymidine incorporation approximately 2.5-fold compared to control cultures, whereas in combination with dexamethasone (10 nM), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [3H]-thymidine incorporation occurring at approximately 1 nM for EGF and insulin and at 100 micrograms ml-1 for FGF. Binding of 125I-EGF to HCF reached maximum after 2 hr at 37 degrees C and was specific, saturable, and of high affinity (half saturation at 1 nM). (ABSTRACT TRUNCATED AT 400 WORDS)

Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis.

2-Deoxy-D-glucose transport by Rhodotorula glutinis is an active process. The intracellular concentration of free deoxyglucose after 15 min incubation of Rhodotorula cells with this sugar was 230 times the extracellular concentration. Although cell extracts at this time contained more 2-deoxy-D-glucose 6-phosphate than deoxyglucose, pulse-labelling experiments demonstrated that deoxyglucose is transported as the free sugar and subsequently phosphorylated. After transport, Rhodotorula cells metabolize deoxyglucose. The major metabolites during 30-90 min incubations were determined to be 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucitol, 2-deoxy-D-gluconate and 2,2'-dideoxy-alpha, alpha'-trehalose. Rhodotorula glutinis also degrades deoxyglucose to CO2. The concentrations of intermediates in this pathway were too low to detect and resolve in extracts of control cells. In 2,4-dinitrophenol-poisoned cells, however, it appears that deoxyglucose degradation is restricted largely to loss of C-1 as CO2 and it was possible to identify 1-deoxy-D-ribulose 5-phosphate as an intermediate presumably arising from metabolism of deoxyglucose by the oxidative portion of the hexose monophosphate pathway.

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis.

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.