Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer.

BACKGROUND: EMT or transformation to the mesenchymal phenotype plays an important role in tumor invasion and metastasis. In vitro data suggest that mesenchymal transformation may correlate with the activation of PI3 kinase and Ras/Erk pathways. We investigated the expression of EMT markers (low E-cadherin, high fibronectin, and vimentin) and their association with p-Erk in resected pancreatic cancer. METHODS: Clinical data/surgical specimens from 34 consecutive pancreatic cancer patients (pts) who underwent pancreatectomy were included. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues using monoclonal antibodies against vimentin, fibronectin, E-cadherin, and p-Erk. The results were correlated with clinicopathological parameters and survival. Survival analysis (log-rank test, Cox proportional hazard model), categorical data analysis (Pearson's chi-square, Fisher's exact test) and Kendall's tau were performed at a significance level of 0.05. RESULTS: The patient population was formed from 13 males and 21 females, with a median age of 66 years (range 38-84 years); American Joint Committee on Cancer (AJCC) stage 1 (n = 2), 2 (n = 27), 3 (n = 5); histological grade 1 (n = 4), 2 (n = 13), 3 (n = 16), 4 (n = 1). Median survival was 15 months (95% CI: 11-24 months). Fibronectin overexpression correlated with the presence of vimentin (p = 0.0048) and activated Erk (p = 0.0264). There was a borderline association of fibronectin with worsening grade (p = 0.06). A negative association between vimentin and E-cadherin was noted (p = 0.0024). Increased fibronectin or vimentin and decreased E-cadherin correlated with poor survival. CONCLUSION: EMT is associated with poor survival in surgically resected pancreatic adenocarcinoma. A correlation between activated Erk and fibronectin was identified that may open avenues for targeted therapy for this subgroup.

Recombinant transforming growth factor type beta 3: biological activities and receptor-binding properties in isolated bone cells.

We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.

Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line.

Two types of growth-modulating factors were derived from the serum-free conditioned media of a human rhabdomyosarcoma cell line, A673. One type, Mr 18,000 to 22,000, competes for binding to epidermal growth factor receptors and enhances the growth of normal and tumor cells in soft agar. It has all of the biological properties ascribed to transforming growth factor type alpha (TGF alpha). A673 cells also produce factors which inhibit the growth of human tumor cells in soft agar and in monolayer cultures. These tumor cell growth-inhibiting factors (TIFs) are acid- and heat-stable peptides. The major TIF activities have molecular weights in the ranges of greater than 28,000, 18,000 to 22,000, 10,000 to 16,000, and 5,000 to 10,000 and do not possess the antiviral activity associated with interferon. Partially purified preparations of TIF-1 (Mr 10,000 to 16,000) inhibit the growth of all human tumor cell lines tested and stimulate the growth of normal human fibroblasts and epithelial cells in monolayer cultures. The growth of human lung carcinoma A549 cells in soft agar, which was enhanced by treatment with TGF alpha from A673-conditioned media, was inhibited by treatment with TIF-1 derived from the same media. The ratio of the two types of tumor cell-derived, growth-modulating factors (TIFs and TGF alpha), which are antagonistic in their biological effects, may determine the capacity of tumor cells for anchorage-independent growth.

Two distinct tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line.

Tumor cell growth-inhibiting factors (TIFs) have been shown to inhibit the growth of tumor cell lines in culture. TIF-1, the first TIF to be described, is a low-molecular-weight, acid- and heat-stable polypeptide with no antiviral activity. A second class of TIFs (TIF-2) has now been isolated from the conditioned media of a human rhabdomyosarcoma cell line and partially purified by polyacrylamide gel filtration, cation exchange, and reverse-phase high-pressure liquid chromatography. Partially purified preparations of TIF-2 inhibit the growth of a variety of human tumor cells in soft agar and monolayer cultures and are mitogenic for normal human and mouse cells. TIF-2 has no antiviral activity. The growth-inhibitory effects of TIF-2 are reversible when the affected cells are no longer exposed to the factor. Although both TIF-1 and TIF-2 are obtained from the same source, they can be distinguished by their molecular weight, heat lability, elution pattern from reverse-phase high-pressure liquid chromatography, and their effect on the growth of mink lung epithelial cells. The growth of a human tumor cell variant, selected for resistance to growth inhibition by TIF-1, is inhibited by TIF-2. TIFs may therefore be a family of related polypeptides which selectively inhibit the growth of tumor cells.

The effects of transforming growth factor beta 3 on the growth of highly enriched hematopoietic progenitor cells derived from normal human bone marrow and peripheral blood.

The effects of transforming growth factor beta 3 (TGF-beta 3) on growth in semisolid cultures of enriched hematopoietic progenitors derived from normal human marrow and blood were evaluated. Conditioned media from the Mo-T cell line (MoCM) were the source of colony-stimulating factors used to optimally stimulate primitive progenitors. To assess whether a proportion of granulocyte/monocyte (GM) progenitors were prevented from cycling, all sizes of GM aggregates were evaluated from 3 to 20 days. The activity of TGF-beta 3 on the growth of erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) was similar to that observed for TGF-beta 1. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or 72 h after initiation of culture, did not significantly affect the total number of marrow GM aggregates at 3, 7, 14, and 20 days, but TGF-beta 3 (1,000 pmol/liter), added initially, reduced the total number of blood GM aggregates. This suggests that some blood GM progenitors might be blocked from cycling but that the great majority of marrow GM progenitors are not blocked. Whether TGF-beta 3 (10, 100, and 1,000 pmol/liter) was added initially or after 72 h of stimulation by MoCM, there was a dose-dependent reduction of marrow and blood GM colony size even when the total number of colonies was unaffected. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or at 72 h, reduced in a dose-dependent manner the size of marrow and blood-derived BFU-E. TGF-beta 3 (1,000 pmol/liter) was more likely to reduce the total number of marrow and blood BFU-E, and this increased sensitivity of the erythroid lineage may prevent the development of this population in colonies derived from multipotential colony-forming unit-granulocyte/erythroid/monocyte (CFU-GEM). The results suggest that the main effect of TGF-beta 3 and TGF-beta 1 is to slow the rate of proliferation of hematopoietic progenitors rather than to prevent them from beginning proliferation. This results in a reduction in colony size which prevents the identification of primitive versus mature progenitor on the basis of standard criteria of colony size.

Prevention of chemotherapy-induced ulcerative mucositis by transforming growth factor beta 3.

Mucositis is a common, dose-limiting complication in patients receiving cancer chemotherapy, which appears to be a consequence of the rate of epithelial proliferation. The beta transforming growth factors have been shown to be negative regulators of epithelial cell proliferation. Here we show that transforming growth factor beta 3 administration reduced proliferation of oral epithelium in vitro and in vivo. Topical application of transforming growth factor beta 3 to the oral mucosa of the Syrian golden hamster prior to chemotherapy significantly reduced the incidence, severity, and duration of oral mucositis, reduced chemotherapy-associated weight loss, and increased survival.

Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase.

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.

Regulation of the levels of three transforming growth factor beta mRNAs by estrogen and their effects on the proliferation of human breast cancer cells.

Transforming growth factor (TGF) beta is a potent regulator of cell proliferation and may play a role in breast cancer cell growth. We have evaluated the regulation of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs by 17 beta-estradiol (E2) and 4-hydroxytamoxifen (MOH) in estrogen receptor-positive (ER(+)) MCF-7 and estrogen receptor-negative (ER(-)) MDA-MB-231 human breast cancer cells. We also determined the effect of TGF beta 1, TGF beta 2, and TGF beta 3 on the proliferation of these cells. Cells were deprived of estrogen before the addition of hormones, and mRNA was measured by Northern blot analysis. We found that MCF-7 cells expressed mRNAs of all three TGF beta species. Treatment of MCF-7 cells with 10(-10) M E2 for 7 days resulted in a dramatic decrease in the TGF beta 2 and TGF beta 3 mRNA levels, but not in the TGF beta 1 mRNA level. MOH was found to block these effects. In addition, the regulation of TGF beta 2 and beta 3 gene expression occurs at both transcriptional and post-transcriptional levels. There is an inverse correlation between E2-induced growth and levels of TGF beta 2 and TGF beta 3 mRNA. In contrast to MCF-7 cells, MDA-MB-231 cells expressed TGF beta 1 and TGF beta 2 mRNAs but TGF beta 3 mRNA was not detected, and the TGF beta 1 and TGF beta 2 mRNAs were not regulated by estrogens or antiestrogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron spin resonance evidence for vertical asymmetry in animal cell membranes.

Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid. Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C. Arrhenius plots of log hH/hP vs. 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively. Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed. Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used. The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO). Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C. Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C. When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed. The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer. In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data.

Regulation of Glutamine Transport in Escherichia coli.

The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate.

Photoreactive probes for high resolution mapping of membrane proteins.

The preparation and characterization of a novel series of radioactively labeled membrane probes is described. These probes are carbohydrate derivatives of fatty acids which contain a photosensitive azide moiety at a specified distance along the alkyl chain. The function of the carbohydrate group is to restrict the azide function to the outer surface monolayer of sealed membrane systems. The azide probes have been used in several well-characterized membrane systems including erythrocyte ghosts, membrane-enveloped viruses, and artificial vesicles. Upon activation, the probes attach to integral proteins to form a stable, covalent complex which may be extracted and identified. The activation protocol is outlined and some of the preliminary results are discussed.

Identification and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains.

The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown.

Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.

Characterization of the three mammalian transforming growth factor-beta (TGF-beta) isoforms, TGF-beta 1, -beta 2, and -beta 3, indicates that TGF-beta 3 is somewhat more potent (ED50 = 0.5 pM versus 2 pM) than TGF-beta 1 and TGF-beta 2 as a growth inhibitor of the Mv1Lu mink lung epithelial cell line. In the fetal bovine heart endothelial (FBHE) cell line, however, TGF-beta 1 and -beta 3 are at least 50-fold more potent than TGF-beta 2 which is a very weak growth inhibitor (ED50 greater than or equal to 0.5 nM). Thus, as growth inhibitors, TGF-beta 1 and -beta 3 resemble each other more than TGF-beta 2. The presence of serum alpha 2-macroglobulin in the FBHE cell assays decreases the biological potency of TGF-beta s, in particular TGF-beta 2. This effect of alpha 2-macroglobulin, however, is not sufficient to explain the low responsiveness of FBHE cells to TGF-beta 2. Evaluation of the role of TGF-beta receptors as determinants of cell-specific responsiveness to TGF-beta isoforms indicates that TGF-beta 1, -beta 2, and -beta 3 have similar affinity for the membrane proteoglycan, betaglycan. They differ, however, in their ability to bind to receptor types I and II which are implicated in TGF-beta signal transduction. TGF-beta 1 is similar, albeit not identical, to TGF-beta 3 and much more potent than TGF-beta 2 as a competitor for binding to the overall population of receptors I and II in all cell lines tested. A subset of receptors I and II has been identified in Mv1Lu cells which has high affinity for TGF-beta 2 (KD approximately 10 pM) and binds this factor at concentrations that are biologically active in Mv1Lu cells. This receptor subset could not be detected in FBHE cells, suggesting that cell-specific differences in the level of high affinity of TGF-beta 2 receptors may lead to cell-specific differences in responsiveness to this isoform. Thus, despite their structural and biological similarities, TGF-beta 1, -beta 2, and -beta 3 diverge in their ability to bind to receptors in a manner that correlates with their potency as growth inhibitors.

Identification of another member of the transforming growth factor type beta gene family.

We report here the complete amino acid sequence of another member of the type beta transforming growth factor gene family, deduced from the nucleotide sequence of three overlapping cDNA clones. The C-terminal 112 amino acids share approximately 80% sequence identity with type beta 1 and beta 2 transforming growth factors, with many of the remaining differences being conservative substitutions. By analogy to type beta 1 and type beta 2 transforming growth factors, we predict the protein to be synthesized as a 412 amino acid precursor that undergoes proteolytic cleavage to produce the mature polypeptide.

Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673.

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.

Umbilical cord transforming growth factor-beta 3: isolation, comparison with recombinant TGF-beta 3 and cellular localization.

The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.

Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice.

Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED(50) = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED(50) = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.