Divergent cell cycle kinetics underlie the distinct functional capacity of mucosal T cells in Crohn's disease and ulcerative colitis.

BACKGROUND: Different abnormalities of T cell effector function distinguish Crohn's disease (CD) from ulcerative colitis (UC). Because cell cycling determines effector function, pathogenic events in CD and UC may depend on cell cycle changes unique to each condition. METHODS: Cell cycle kinetics, cycle regulatory molecule expression, apoptosis, caspase and telomerase activity, and cellular expansion were evaluated in CD2 and CD3 activated control, CD, and UC lamina propria T cells. RESULTS: Compared with normal cells, CD T cells cycle faster, express increased phosphorylated Rb and decreased phosphorylated p53 levels, display less caspase activity but more telomerase activity, die less, and undergo vigorous cellular expansion. In contrast, UC T cells cycle slower, express normal levels of phosphorylated Rb and p53, display more caspase activity but have no telomerase activity, die more, and have a limited capacity to expand. CONCLUSIONS: T cell cycle abnormalities in CD indicate a state of hyperreactivity compatible with loss of tolerance, but a hyporeactive state compatible with anergy in UC. Thus distinct and divergent T cell cycle characteristics underlie the pathogenesis of the two main forms of inflammatory bowel disease.

Cyclosporin A effects during primary and secondary activation of human umbilical cord blood T lymphocytes.

OBJECTIVE: Cyclosporin A (CsA), effective in prophylaxis and treatment of graft-vs-host disease (GVHD) after human allogeneic transplantation, blunts T-cell responses by inhibiting nuclear factor of activated T cells-1 (NFAT1) activation. This laboratory has shown that NFAT1 protein expression is severely reduced in human UCB (umbilical cord blood) T cells. Since UCB is increasingly used as a hematopoietic stem cell source in allogeneic transplantation, it is important to determine whether CsA sensitivity in UCB differs from that of adult T cells. METHODS: Surface flow cytometric analysis, intracellular cytokine staining, flow cytometric analysis of cell death, and thymidine incorporation were used in this study to determine T-cell activation and effector functions during primary and secondary stimulation in the presence of CsA. RESULTS: Although we observed differential CsA sensitivity of T-cell activation marker (CD69, CD45RO, CD25) upregulation comparing UCB and adult, we did not observe any significant difference in CsA sensitivity of T-cell effector functions. Importantly, we observed reduced IFN-gamma and TNF-alpha expression in UCB T cells both in primary and secondary stimulation, as well as increased rates of activation-induced cell death (AICD). CONCLUSION: Thus, our studies do not support the previous hypothesis that reduced GVHD observed after UCB transplantation is attributable to increased CsA sensitivity of UCB T cells. Rather, reduced UCB T-cell cytokine production and increased AICD may be important cellular mechanisms underlying these favorable rates of GVHD in UCB transplant recipients.

Stoichiometry of immunocytochemical staining reactions.

This chapter reviewed and presented my biases about the factors that affect our ability to make quantitative measurements of epitope (for this chapter, equal to a protein) with monoclonal antibodies in a flow cytometric system. The discussion has illustrated that the chemical structure of the permeabilized cell and the affinity and specificity of the antibody are the "two" factors that are important. Pursuit of this discipline would be significantly enhanced with highly specific antibodies with a high affinity--higher than we have so far observed with the antibodies against cell cycle regulatory proteins. When moving to multiparametric space, optical systems that mask off interbeam cross talk, primary labeled antibodies, and rigorous use of fluorescence compensation is essential for the highest quality work.

TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression.

Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors.

A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype.

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

Molecular quantification of cell cycle-related gene expression at the protein level.

BACKGROUND: Immunofluorescence cytometry of antigen and DNA content provides relative measurements of the cell cycle phase distribution of a specific epitope. Measurement of correlated expression of epitopes on signaling and regulatory proteins will be useful in the study of the complex pathways involved in cell cycle regulation and carcinogenesis. However, to formulate regulatory pathway models, measurements of molecules per cell would be more useful than relative measurements of intensity. Here, we report on a system in which the relationship between molecules and fluorescence is determined for a reference set of cell lines that are then used to directly calculate the number of molecules for unknowns. To demonstrate the process, we calculated the cell cycle phase distribution of SV40 large T antigen (Tag) in the reference cells. METHODS: A set of cell line clones expressing different levels of Tag were isolated. Quantitative Western blots of these cells and purified, recombinant Tag were performed. Cells from the same sample were stained and analyzed by flow cytometry for Tag and DNA. The relationship between molecules and fluorescence was established and calculations were performed for the phase distributions of Tag. RESULTS: The five cell lines had 0.11, 0.27, 1.06, 2.44, and 2.63 x 10(6) molecules of Tag per cell, determined by Western blot. The average coefficient of variation was 10.6%. The relationship of molecules to fluorescence fit a linear equation (r(2) = 0.96) over the range, 0.11 - 2.63 x 10(6) molecules, however, the same equation did not fit the relationship between 0 molecules, defined by isotype staining controls, and the lowest expressing cell line. To calculate the phase distributions of molecules in the lowest cell line, a second linear equation from 0 to 110,000 molecules was used. CONCLUSIONS: This work describes a system where fixed cells expressing various levels of a target antigen quantified by Western blots can be used to standardize flow cytometric measurements of gene expression in absolute terms.

Reduced NFAT1 protein expression in human umbilical cord blood T lymphocytes.

Umbilical cord blood (UCB) stem cells from related and unrelated allogeneic donors have emerged as novel treatment for patients with hematologic malignancies. The incidence and severity of acute graft-versus-host disease (GVHD) after UCB transplantation compares favorably with that observed in recipients of matched unrelated donor allogeneic grafts, but remains a major cause of morbidity and mortality. It has been shown that stimulated lymphocytes from UCB have reduced production of cytokines including interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), which play a role in GVHD pathophysiology. We investigated the molecular mechanisms underlying this reduced cytokine production by analyzing expression of nuclear factor of activated T cells-1 (NFAT1) in UCB T cells. We detected no constitutive expression of NFAT1 protein in unstimulated UCB T cells compared with adult T cells. Moreover, although NFAT1 expression in UCB T cells was upregulated after prolonged (40 hours) T-cell stimulation, it was only partially upregulated when compared with adult controls. Our observation of minimal NFAT1 expression after stimulation correlated with reduced cytoplasmic IFN-gamma and TNF-alpha production in UCB T cells studied simultaneously. Reduced NFAT1 expression may blunt amplification of donor UCB T-cell alloresponsiveness against recipient antigens, thereby potentially limiting GVHD incidence and severity after allogeneic UCB transplantation.

Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy.

BACKGROUND: Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2). METHODS: Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene. RESULTS: Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells. CONCLUSIONS: A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.

A new human prostate carcinoma cell line, 22Rv1.

A cell line has been derived from a human prostatic carcinoma xenograft, CWR22R. This represents one of very few available cell lines representative of this disease. The cell line is derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft. Flow cytometric and cytogenetic analysis showed that this cell line represents one hyper DNA-diploid stem line with two clonal, evolved cytogenetic sublines. The basic karyotype is close to that of the grandparent xenograft, CWR22, and is relatively simple with 50 chromosomes. In nude mice, the line forms tumors with morphology similar to that of the xenografts, and like the parental CWR22 and CWR22R xenografts, this cell line expresses prostate specific antigen. Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis. Growth is stimulated by epidermal growth factor but is not inhibited by transforming growth factor-beta1.

Simultaneous detection of cyclin B1, p105, and DNA content provides complete cell cycle phase fraction analysis of cells that endoreduplicate.

BACKGROUND: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations. METHODS: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection. RESULTS: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated. CONCLUSIONS: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.

Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data.

BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.

Cell cycle analysis of retroviral vector gene expression during early infection.

To examine the pattern of retroviral vector gene expression during early stages of infection, a Moloney murine leukemia virus (M-MuLV)-based vector that transcribes the simian virus 40 (SV40) large T antigen (Tag) gene from the viral long terminal repeat (LTR) was used to infect proliferating rodent fibroblasts. At various times after infection, cells were fixed and stained for Tag by indirect immunofluorescence, and for DNA using propidium iodide. Tag immunofluorescence and DNA content were both quantified by flow cytometry. The results showed that Tag expression was first detected exclusively in the late G1 and early S phases of the cell cycle, approximately 12 h postinfection. The infection was synchronous in that Tag-expressing cells detected at 12 h, in late G1 and early S, moved as a discrete population through S phase, into the G2 + M phases of the cell cycle and then back into G1 during the next 8-10 h. The presence of a synchronous Tag-expressing cell population suggests that, at time of infection, cells in certain phases of the cell cycle were more susceptible to infection than cells in other phases. This may be related to synchronizing events that must occur before viral genes are expressed in infected cells; one such event may be integration of viral DNA into cellular chromosomes (i.e., provirus formation) that requires cells to pass through an M phase.

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.

TGF-beta 1 perturbation of the fibroblast cell cycle during exponential growth: switching between negative and positive regulation.

We have demonstrated previously that SV40 T antigen and serum regulate the length of G1 in exponentially growing NIH-3T3 cells in part by inhibiting density dependent negative cell cycle regulation. In these studies it was suggested that T antigen positively regulated G1 in a density independent manner as well. In this report we show that, 24 h after treatment, TGF-beta 1 perturbs the cell cycle of exponentially growing fibroblasts in a manner similar to T antigen. However, prior to 24 h, TGF-beta 1 produced a negative response, elongating the G1 phase of the cell cycle that was followed by a positive response, both of which were density independent. This biphasic response was measured between 0 and 12 h post-treatment and was relative to responses from serum. This switch from an early inhibitory effect to a late stimulatory effect was associated with changes in Rb phosphorylation, the timing and magnitude of which indicated that Rb may be directly regulating TG1 rather than reporting changes in the population. This is further substantiated by abrogation of the inhibitory effect by expression of wild-type SV40 T antigen and retention of the effect in cells that express an Rb-binding mutant of T antigen (K1). The biphasic regulatory effects of TGF-beta 1 were also displayed in WI-38 and IMR-90 human fibroblasts. This suggests that this biphasic effect is a property of fibroblasts.

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.

Stepwise transformation of astrocytes by simian virus 40 large T antigen and epidermal growth factor receptor overexpression.

We have investigated the transformed phenotype of neonatal mouse cortical astrocytes immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene. Expression of T antigen was driven by the Moloney murine leukemia virus long terminal repeat. Cell lines were selected based on coexpression of neomycin resistance, which provides a selection method believed to be unbiased for transformation state. Astrocyte cell lines derived in this manner express T antigen over a relatively narrow range (approximately 4-fold), are contact inhibited, are able to enter a quiescent state in the presence of growth factors, and do not readily form colonies in soft agar. Compared to mortal astrocytes, the population growth rate is increased 3-fold, saturation densities are 4-fold higher, and the genome is relatively unstable as measured by the presence of DNA-aneuploid stem lines and by changes in DNA ploidy over time. However, changes in transformation phenotype occur at a low rate, making the cell lines amenable to experimentation. Most often, the growth phenotype remained unchanged during months of culture. Transfection of an epidermal growth factor receptor (EGFR) gene was used to generate a subline that was conditionally transformed (colony formation in soft agar was dependent on transforming growth factor alpha). v-raf transfection was used to generate constitutive transformation. Thus, these cell lines appear to be excellent experimental models for progressive transformation. With them, untested hypotheses of brain tumor progression derived from human genetic studies may be tested experimentally.

Immortalization of epithelial cells.

The methodologies for isolating cell lines have become very powerful, particularly in terms of retaining differentiated features of the parent cells. Cell lines can be developed from primary or early passage cells as well as from transgenic animals that carry an immortalizing gene. Cell lines from epithelia have been selected for their polar orientation, tight junction formation, and expression of differentiated markers or functions. These cell lines provide useful models for studying cell biology of specific tissues, tumorigenicity, genetic abnormalities, or to help screen for effective methods of gene therapy.

Transforming growth factor beta regulation of epidermal growth factor receptor in ectocervical epithelial cells.

Transforming growth factor beta (TGF beta) is a pluripotent modulator of cell function and an important suppressor of cervical epithelial cell proliferation. In the present study, we examine the effects of TGF beta 1 on the level and activity of the epidermal growth factor receptor (EGFR) in HPV-16 immortalized cervical epithelial cells. In ECE16-1 cells, increased EGFR levels are observed within 24 h after initiation of TGF beta 1 treatment and levels continue to increase with time. This increase is correlated with a TGF beta 1-dependent decrease in proliferation rate. Scatchard analysis indicates that the population of EGFR sites induced by TGF beta 1 have a low affinity for EGF (Kd = 4.08 nM) compared to the receptors present prior to TGF beta 1 treatment (Kd = 0.3 and 1.6 nM). TGF beta 1 treatment also reduces EGFR kinase autophosphorylation activity. Cell cycle studies indicate that TGF beta 1-treated cells arrest in the G1 phase of the cell cycle and that regulation of EGFR level was independent of cell cycle stage in both TGF beta 1-treated and untreated cells. However, EGFR level was related to the G1 phase time. Parallel studies indicate that a TGF beta 1-dependent increase in p53 level is also correlated with increased time spent in G1. These results suggest that TGF beta 1 inhibition of ECE16-1 cell proliferation may act both by the replacement of high affinity/high kinase activity EGFR sites with low affinity/low kinase activity EGFR sites and a p53-mediated cell cycle arrest.