Ligand induced internalization of epidermal growth factor receptors by A431 cells decreases at high cell densities in culture.

Internalization of epidermal growth factor (EGF) receptors by human A431 epidermoid carcinoma cells was studied at various culture densities. The extent of EGF receptor internalization was measured by quantitation of internalized 125I-EGF during incubation at 37 degrees C for 30 min. When cell culture density was below 1 x 10(5) cells/cm2 receptor internalization was active; 30-40% excess moles of ligand over the moles of surface EGF receptors were internalized during this period. However, when culture density increased to above 1.5 x 10(5) cells/cm2 receptor internalization became less extensive, as only 30-50% of ligand bound to the cell surface underwent internalization during 30 min incubation. In parallel with this reduction in receptor internalization, the degradation rate of 35S-methionine labeled EGF receptors was reduced at a high culture density. In contrast with this regulation of receptor internalization, the affinity of EGF receptors for the ligand increased as culture density increased. The extent of EGF-dependent receptor phosphorylation was found to be constant at all culture densities tested. Thus, the observed low level of receptor internalization at high culture densities was not attributable to lower responsiveness of receptors to EGF. These data suggest the presence of an as yet unidentified cell density-dependent mechanism for regulating receptor internalization in cultured A431 cells.     

Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. III. Effects of chronic exposure to epidermal growth factor on growth, differentiation, and collagen biosynthesis

Studies have been performed to evaluate the effects of chronic exposure to epidermal growth factor (EGF) on a cell line (FRLE cells) established from the fetal rat lung type II alveolar epithelial cell. Chronic exposure to EGF enhanced proliferation and altered the culture morphology at the light microscopic level. At the ultrastructural level, chronic exposure to EGF inhibited the expression of lamellar body-like structures that occurs in the absence of EGF. Estimations of total protein and collagen production indicated that chronic exposure to EGF suppressed collagen production without significantly altering total protein synthesis. Quantitative evaluation of the genetic types of collagen (types I, III, IV, and V) produced by FRLE cells revealed decreased production of each collagen type in cultures chronically exposed to EGF. However, the magnitude of the decrease differed for each collagen type, with type III collagen synthesis being suppressed to the greatest extent. Additionally, chronic exposure to EGF resulted in enhanced secretion of types I, III, and IV collagen molecules and an increase in the ratios of type I-homotrimers to type I-heterotrimers and of type V-homotrimers to type V-heterotrimers. These findings demonstrate that chronic exposure to EGF selectively alters collagen expression in a cell line established from the fetal type II pneumocyte.     

Enhancement of disaturated phosphatidylcholine synthesis by epidermal growth factor in cultured fetal lung cells involves a fibroblast-epithelial cell interaction

Epidermal growth factor (EGF) increases the rate of choline incorporation into disaturated phosphatidylcholine in cultured fetal rat type II cells via an indirect mechanism. Whereas-EGF has no effect on the rate of disaturated phosphatidylcholine synthesis when added directly to type II pneumocytes, the growth factor is effective if it is present during preliminary conditioning of the media by lung fibroblasts. This effect is concentration dependent with a maximal effect at 20 ng/ml. When lung fibroblasts are incubated with both glucocorticoids and EGF, there is no significant effect of the growth factor over and above that seen with the steroid alone. This suggests that the two agents might act via a similar mechanism. This is supported by the observation that each inducer leads to the production by lung fibroblasts of a stimulatory factor that has a similar, if not identical, chromatographic elution profile. We conclude that EGF may contribute significantly to the normal onset of lung maturation by elaborating a fibroblast-derived factor that stimulates phosphatidylcholine synthesis in type II pneumocytes.     

Separation of transforming growth factors TGF alpha and TGF beta during chromatographic purification of extracts from mouse C-234 tumors

Polypeptide transforming growth factors: TGF alpha and TGF beta were isolated and separated from acidic ethanol extracts of mouse C-243 tumors. The purification of the acid-soluble extract was achieved by Bio-Gel P-60 filtration chromatography, followed by CM-Sepharose CL-6B ion exchange, Bio-Gel P-10 filtration, and dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At the Bio-Gel P-10 purification step, TGF alpha was separated from TGF beta. TGF alpha stimulated mouse Balb/c-3T3, rat NRK-49F and human A549 cells to form colonies in soft agar, and competed with 125I-labeled epidermal growth factor (EGF) for binding to human placenta membrane receptors. Over 40,000-fold SDS-PAGE-purified TGF beta had an Mr of 25,000. Unlike TGF alpha, TGF beta stimulated the clonal growth of NRK fibroblasts only in the presence of the suboptimal amounts of EGF (0.5 ng/ml). TGF beta significantly inhibited the anchorage-independent growth of malignant human lung carcinoma A549 cells, and in the radioreceptor assay with 125I-EGF it had no affinity to EGF receptors.     

A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. METHODS: The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. CONCLUSION: Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.     

Keratinocyte growth factor ameliorates radiation- and bleomycin-induced lung injury and mortality.

Keratinocyte growth factor (KGF) is a growth factor for type II pneumocytes. Type II pneumocyte hyperplasia, a common reaction to lung injury, has been postulated to play an important role in lung repair. The potential protective effect of KGF was therefore studied in rat models of radiation- and bleomycin-induced lung injury. Intratracheal instillation of KGF (5 mg/kg) 72 and 48 hours before 18 Gy of bilateral thoracic irradiation did not significantly improve survival, although histology showed less pneumonitis and fibrosis in KGF-pretreated as compared with control-irradiated rats. Intratracheal pretreatment with KGF in rats receiving intratracheal bleomycin (2.5 U) improved survival at 3 weeks to 100% (20/20 rats) from 40% (8/20 rats) in controls. All KGF-pretreated rats receiving bleomycin were well at 3 weeks and without histological evidence of pulmonary fibrosis whereas the 8 surviving control rats exhibited severe respiratory distress. Finally, in the most lethal challenge to the lung, rats pretreated with intratracheal KGF or saline were challenged with a combination of bleomycin (1.5 U) and bilateral thoracic irradiation (18 Gy). KGF-pretreated rats did not begin to die or show signs of respiratory distress until 7 weeks, whereas all saline-pretreated control rats receiving radiation and bleomycin died within approximately 4 weeks with severe respiratory distress and weight loss. In conclusion, radiation- and bleomycin-induced pulmonary injury and respiratory death are ameliorated by KGF pretreatment, suggesting a protective role for KGF-induced type II pneumocyte proliferation in lung injury.     

Donor-derived type II pneumocytes are rare in the lungs of allogeneic hematopoietic cell transplant recipients

Lung injury is a common cause of death and disability. Stem cell-related therapies are widely viewed as offering promise for people suffering from various types of pulmonary diseases, and gender-mismatched bone marrow transplant recipients serve as natural populations in which to study the role of bone marrow-derived stem cells in recovery from pulmonary injury. We evaluated the extent of lung repopulation by type II pneumocyte descendents of adult bone marrow-derived stem cells in allogeneic hematopoietic cell transplant recipients. Recut sections were obtained from five lung biopsy specimens and autopsy lung tissues from four female recipients of transplanted mobilized peripheral blood stem cells or bone marrow from male donors. Sequential immunohistochemistry and fluorescence in situ hybridization was performed on each section to evaluate for Y-chromosome-containing type II pneumocytes. A single Y-chromosome-containing type II pneumocyte was found in one lung biopsy from one hematopoietic cell transplant recipient. After adjustment for the effects of incomplete nuclear sampling, this pneumocyte represented 1.75% of all type II pneumocytes in the biopsy sample. There was no evidence of polyploidy to suggest cell-to-cell fusion. No donor-derived type II pneumocytes were found in samples from the other three patients. In conclusion, repopulation by bone marrow-derived stem cells or their progeny occurs at a low frequency in the lungs of hematopoietic cell transplant recipients. Conversely, proliferation by local stem cell populations appears to be more important for recovery from alveolar injury.     

Zinc-dependent cytoadherence of Legionella pneumophila to human alveolar epithelial cells in vitro

Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.     

Stimulation of stromal cell growth by normal rat urothelial cell-derived epidermal growth factor

Primary cultured adult rat urothelial (RU) cells caused increased thymidine incorporation in rat bladder stromal (RS) cells in a coculture system. The concentrated conditioned medium derived from RU cell culture (CM-RU) also stimulated the growth of RS cells, and induced anchorage independent growth of NRK-49F cells. Since these activities were heat and acid resistant, we investigated whether epidermal growth factor (EGF) and/or transforming growth factors were the humoral factor(s) responsible. The immunodiffusion analysis of CM-RU gave a positive precipitin line with rabbit anti-rat EGF IgG but not with rabbit anti-rat transforming growth factor-alpha antibodies. The anti-rat EGF IgG inhibited CM-RU-stimulated thymidine incorporation into RS cells, whereas normal rabbit IgG did not. By immunofluorescent technique using rabbit anti-rat EGF antibodies, immunoreactive EGF was demonstrated in RU cells and the urothelial of cryoinjury-induced reparative hyperplasia. Immunofluorescent technique also demonstrated the presence of EGF receptors on the cell membrane of RU and RS cells, basal cells of normal rat urothelium, and cells of whole epithelial layers of reparative hyperplasia. These data strongly suggest that EGF or an EGF-like substance produced by RU cells and released into medium stimulates the growth of RS cells possibly mediated by EGF receptors.     

Anti-bombesin monoclonal antibodies modulate fetal mouse lung growth and maturation in utero and in organ cultures

Fetal pulmonary neuroendocrine cells (PNECs) contain abundant gastrin-releasing peptide (GRP, mammalian bombesin-like peptide [BLP]). Previously, addition of bombesin resulted in increased fetal lung growth and maturation in utero and in organ cultures. A monoclonal antibody (mAb) to bombesin (2A11) blocked baseline automaturation of lung organ cultures in serum-free medium. In the present study, we analyze lung development following daily in utero administration of 2A11 from gestational days 15–18. Fetal lung treated with 2A11 and then harvested on day 18 demonstrated a dose-dependent decrease in surfactant phospholipid synthesis compared to controls treated with MOPC, an unreactive mAb. However, 2A11-treated fetal lung harvested on day 17 showed paradoxical increases in ³H-choline incorporation into saturated phosphatidylcholine, ³H-thymidine incorporation into DNA, and relative numbers of differentiated type II pneumocytes. In serum-containing day 17 lung organ cultures, 2A11 stimulated choline and thymidine incorporation. Since epidermal growth factor (EGF) is the only agent besides bombesin known to stimulate both fetal lung growth and maturation, we added EGF to serum-free cultures and reconstituted the stimulatory effects. A murine EGF receptor mAb (ERA) blocked 2A11-induced lung growth and maturation in serum-containing cultures, and this effect was overcome by adding EGF. In vivo, ERA also blocked stimulatory effects of 2A11 in fetal lung on day 17. These observations suggest that EGF receptor up-regulation may maintain lung growth and maturation if BLP levels are diminished on day 17. Nonetheless, BLPs appear to be involved in lung maturation on day 18, supporting a role for PNECs in normal lung development. © 1993 Wiley-Liss, Inc.     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

Keratinocyte Growth Factor Decreases Pulmonary Edema, Transforming Growth Factor-Beta and Platelet-Derived Growth Factor-BB Expression, and Alveolar Type II Cell Loss in Bleomycin-Induced Lung Injury

Keratinocyte growth factor (KGF), a potent growth factor for type II pneumocytes and Clara cells, has been shown to prevent the end-stage pulmonary fibrosis and mortality in a rat model of bleomycin-induced lung injury. In this study, protective effects of KGF were explored during the earlier course of bleomycin-induced lung injury by studying protein exudation in alveolar edema fluids, pulmonary expression of transforming growth factor-beta (TGF) and platelet-derived growth factor-BB (PDGF-BB), and changes in type II pneumocytes and Clara cells after i.t. (intratracheal) bleomycin injection following KGF- or saline-pretreatment in rats. Total protein in bronchoalveolar lavage (BAL) fluids after bleomycin injury from KGF-pretreated rats was significantly lower than the levels in saline-pretreated rats. TGF protein in BAL fluids which peaked at day 3 after i.t. bleomycin in saline-pretreated lungs was not significantly increased at any time points in KGF-pretreated rats. PDGF-BB protein in whole lung tissues of KGF-pretreated rats also remained near normal throughout the course after i.t. bleomycin, in contrast to the significant increase in saline-pretreated rats. Numbers of type II pneumocytes and Clara cells in KGF-pretreated lungs after a high dose of bleomycin were close to the normal in intact lungs. At the same dose of bleomycin injury, type II pneumocytes in saline-pretreated lungs were markedly decreased, while the number of Clara cells in these rats was relatively preserved as the pre-injury level. In conclusion, KGF prevents bleomycin-induced end-stage pulmonary injury and mortality probably at least partly by decreasing protein-rich pulmonary edema, protein expression of fibrogenic cytokines TGF and PDGF-BB, and type II cell loss during the course of lung injury.     

Isolation and partial characterization of pneumocin, a novel apical membrane surface glycoprotein marker of rat type II cells

Rat alveolar type II pneumocytes, in situ, label with Maclura pomifera agglutinin (MPA), a plant lectin that recognizes alpha-galactosyl oligosaccharide residues of glycoproteins and glycolipids. To study the glycoproteins recognized by the lectin, MPA lectin affinity chromatography was used to isolate a novel glycoprotein, pneumocin, from type II and whole rat lung cell membranes. Pneumocin isolated from adult rat lungs was a non-disulfide-linked sialoglycoprotein with an Mr of 165 kD. Asparagine-linked oligosaccharides contributed 5 to 10% to the Mr. Two-dimensional chymotryptic peptide maps of pneumocin isolated from whole lung membranes and type II cells were similar. The glycoprotein partitioned in the detergent phase on Triton X-114 phase separation. Murine monoclonal antibodies developed against the purified glycoprotein localized on apical membranes of type II pneumocytes in situ. The antibodies did not label type I cells or lamellar bodies but labeled luminal surfaces of vesicular structures of type II cells. Isolated type II cells labeled with antibodies after 1 d in culture but showed significantly less staining of cells after 4 d of culture. These observations demonstrate that pneumocin is a cell surface sialoglycoprotein marker of type II cells. Western blot analysis of liver and kidney cell membranes suggest that related glycoproteins may also be present in those tissues. The isolation technique and monoclonal antibodies should permit further characterization and functional studies of the glycoprotein.     

Role of inositol triphosphate isomer formation in type II pneumocyte signal transduction.

Adenosine triphosphate (ATP) is a potent agonist of surfactant secretion from type II pneumocytes. The extracellular ATP signal is transduced by both P1- and P2-purinergic pathways, which respectively initiate cyclic adenosine monophosphate formation and phosphatidyl inositol hydrolysis to inositol phosphates (Ins P). We investigated the role of inositol triphosphate (Ins P3) isomer formation in this signal transduction pathway. Primary cultures of rat type II pneumocytes were labeled with 30 microCi [3H]myoinositol/5 x 10(6) cells for 48 h. After preincubation with 10 mM LiCl for 20 min, the cells were stimulated with ATP (10(-4) M) and then were rapidly frozen with liquid N2. The Ins P3 isomers were analyzed by high performance liquid chromatography. A 4-fold increase in Ins 1,4,5 P3 occurred within 2 s after stimulation with ATP, decreased to half maximum by 60 s, and returned close to baseline values by 2 min. In contrast, Ins 1,3,4 P3 did not increase until 15 s, peaked by 60 s with a 4-fold increase, and returned to baseline values by 2 min. Intracellular calcium [( Ca2+]i), measured as Indo-1 fluorescence, also increased 3-fold within 2 s of exposure to ATP (10(-4)M) and fell to a plateau level 25% above baseline values by 10 s. We conclude that Ins 1,4,5 P3 formation and [Ca2+]i release both occur rapidly after exposure of type II pneumocytes to extracellular ATP. We speculate that these early events in type II pneumocyte signal transduction play a role in the initiation of stimulation of surfactant secretion by extracellular ATP.     

Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro

Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.     

Hyperplasia of type II pneumocytes in acute lung injury. Cytologic findings of sequential bronchoalveolar lavage.

The organizing stage of acute lung injury syndrome is characterized, in part, by a reactive hyperplasia of type II pneumocytes. These cells may be recovered in large numbers and in an excellent state of preservation by bronchoalveolar lavage. It was noted recently that such cells in lavage samples may mimic adenocarcinoma. To examine the dynamics of type II pneumocyte shedding in acute lung injury, we studied 62 bronchoalveolar lavage samples from 38 patients with acute onset of the adult respiratory distress syndrome (median age, 54 years). A single lavage was performed in 25 patients, whereas from two to five studies were performed in 13 individuals. The timing of lavage ranged from 1 to 435 days after the onset of respiratory distress. Type II pneumocytes were present in 12 specimens as follows: 6 of 24 (25%) samples from days 1 to 3, 4 of 9 (44%) samples from days 4 to 10, and 2 of 11 (18%) samples from days 21 to 32. Specimens obtained after day 32 never contained such cells. Cytologically, type II pneumocytes may resemble the cells of acute radiation injury. Alternatively, they may be smaller, with an increased nuclear-cytoplasmic ratio, nuclear membrane irregularities, and prominent nucleoli, thus resembling the cells of adenocarcinoma. They may shed singly or in groups. A spectrum of changes from small cells to large, fully hyperplastic type II cells may be seen. Recognition of the morphologic features of type II pneumocytes and careful clinical correlation usually will suffice to prevent a false diagnosis of malignancy. Sequential lavage provides a means to study pulmonary epithelial changes after lung injury.     

Zinc-dependent cytoadherence of Legionella pneumophila to human alveolar epithelial cells in vitro

Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.     

Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.     

Interactions between initiating chemical carcinogens, tumor promoters, and adenovirus in cell transformation

Cell culture systems that respond to the combined action of initiating chemical carcinogens, tumor promoters, and transforming viruses represent useful model systems for studying the complex multifactor nature of the carcinogenic process. We have utilized both secondary rat embryo (2 degrees RE) and a clonal population of established Fischer rat embryo (CREF) cells to study the effect of multiple agents on the process of adenovirus transformation. In the present review we summarize our investigations on the effects of polycyclic aromatic hydrocarbons, tumor promoters, a bee venom polypeptide-melittin (MEL), and the polypeptide hormone epidermal growth factor (EGF) on transformation of rat embryo cells by a temperature-sensitive mutant of human adenovirus type 5 (H5ts125). We also describe the effect of tumor promoters on the progression of the transformed phenotype, ie the temporal acquisition of anchorage-independent growth in adenovirus-transformed clones and the ability of MEL and EGF to elicit effects similar to those of tumor promoters in adenovirus-transformed cells.