A proliferative effect of transforming growth factor-beta1 on a human prostate cancer cell line, TSU-Pr1

Transforming growth factor -beta (TGF-beta) is growth inhibitory to many malignant cells, including prostate cancer cells. The present study reports an unusual observation in that TGF-beta is growth stimulatory to a human prostate cancer cell line, TSU-Pr1. The TSU-Pr1 line is highly aggressive and exhibits a rapid rate of proliferation in culture. These cells underwent further proliferation in response to TGF-beta1. Both type I and II receptors to TGF-beta (TPR-I, TPR-II) are expressed in TSU-Pr1 cells. Activation of a luciferase reporter gene, which contains a TGF-beta response element, confirmed that the TGF-beta receptors in TSU-Pr1 cells were functional. RT-PCR analysis and an ELISA assay determined that TSU-Pr1 cells secreted TGF-beta. In conclusion, TSU-Pr1 cells contain functional TGF-beta receptors but instead of the usual growth inhibition by TGF-beta1, these cells undergo proliferation. The present observation provides a proliferative role of TGF-beta in TSU-Pr1 cells, which may play a part in the aggressive phenotype of these cells and, perhaps other prostate cancer cells.     

Regulation of complement C3 synthesis by interleukin-1 and transforming growth factor-beta in rat non-transformed intestinal epithelial cell line, IEC-6

Intestinal epithelial cells are an important source of many biologically active molecules that modulate immune responses in the mucosa. The purpose of this study was to demonstrate the synthesis of complement C3 component in the rat non-transformed crypt-like intestinal epithelial cell line, IEC-6. Unstimulated IEC-6 cells secreted a low level of C3 protein and showed weak expression of C3 mRNA. The addition of interleukin (IL)-1 beta induced a dose- and time-dependent increase in C3 production. These effects of IL-1 beta were observed at a concentration as low as 0.01 ng/ml and reached a plateau at a concentration of 5 ng/ml. The effects were observed at the mRNA level as early as 6 h after the beginning of incubation. Transforming growth factor (TGF)-beta alone had no effect. However, TGF-beta at low concentrations (0.001-1 ng/ml) enhanced the effect of IL-1 beta in increasing C3 production; this enhancement was not observed at high concentrations (5-10 ng/ml). These effects of TGF-beta were also observed at the mRNA level. The present findings indicate that intestinal epithelial cells are indeed capable of synthesizing complement C3 in response to IL-1 beta and TGF-beta.     

Lck associates with and is activated by Kit in a small cell lung cancer cell line: inhibition of SCF-mediated growth by the Src family kinase inhibitor PP1

At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.     

Type 1 astrocytes influence luteinizing hormone-releasing hormone release from the hypothalamic cell line GT1-1: is transforming growth factor-beta the principle involved?

The possible existence of a humoral communication between glial cells and LHRH-secreting neurons has been studied using the LHRH-secreting GT1-1 cell line and type 1 astrocytes. Two different designs have been adopted: 1) GT1-1 cells were coincubated with purified cultures of type 1 rat astrocytes, and 2) GT1-1 cells were exposed to the conditioned medium (CM) in which type 1 rat astrocytes had been grown for 24 h. LHRH was measured by RIA in the medium of the GT1-1 cell cultures at different time intervals. The data show that short periods (1, 3, and 6 h) of either coculture or exposure to previously frozen CM significantly increase the release of LHRH from the GT1-1 cells. However, more prolonged times of coculture (e.g. 2 and 5 days) or exposure to CM (e.g. 48 h) induce a significant decrease in the amount of LHRH in the medium. The stimulatory effect on LHRH release appears to be specific for type 1 astrocytes (either cortical or hypothalamic), because neither the CM of oligodendrocytes nor the CM of LNCaP cells (a cell line derived from a human prostatic cancer) possess stimulating activities. Heating the type 1 astrocyte-CM to 100 C for 10 min does not eliminate the ability of the CM to significantly increase the release of LHRH from GT1-1 cells at 1, 3, and 6 h. Because of the opposite effects encountered in the short and long term experiments, it was hypothesized that the CM might contain, in addition to LHRH-releasing principle(s), LHRH-degrading properties. Known amounts of standard LHRH were then added to type 1 astrocyte-CM, either untreated or submitted to heating at 100 C for 10 min. The amount of LHRH added to untreated CM decreases progressively; on the contrary, the amount of LHRH added to heated CM remains unchanged. These results confirm that one or more heat-sensitive enzymes able to degrade LHRH may be present in the type 1 astrocyte-CM. As previously mentioned, the experiments reported so far were performed using type 1 astrocyte-CM that had been kept frozen for various periods of time, before being tested for its LHRH-releasing activity. Surprisingly, fresh CM proves to be inactive, whereas heated CM is effective; this suggests that the factor involved might be activated by the two opposite experimental procedures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of apoptosis by transforming growth factor-beta 1 in the rat hepatoma cell line McA-RH7777: a possible association with tissue transglutaminase expression

We report here that transforming growth factor-beta 1 induces cell death in the Morris hepatoma cell line McA-RH7777. We assessed the type of cell death induced by transforming growth factor-beta 1 in this hepatoma cell line on the basis of morphological and biochemical characteristics. Dying cells, which detached from the cell monolayer, showed morphological characteristics of apoptosis (programmed cell death) such as chromatin condensation, nuclear disintegration and cellular fragmentation into clusters of eosinophilic globules. DNA isolated from these cells showed a ladder pattern consisting of multimers of 180 to 190 bp, indicating extensive DNA cleavage into oligonucleosomal units by an endogenous endonuclease. Treatment of the dead cells with detergents and chaotropic agents resulted in formation of insoluble shells, so-called apoptotic bodies, suggesting extensive cross-linking of cell proteins by tissue transglutaminase. Furthermore, increased amounts of cytosolic tissue transglutaminase, which has been recognized as a possible marker of apoptosis, and extensive cross-linking of cytokeratin polypeptides was demonstrated in TGF-beta 1-treated hepatoma cells on immunoblot analysis. These results provide strong evidence that the cell death induced by TGF-beta 1 in McA-RH7777 hepatoma cells is mainly apoptotic. It also suggests that a specific induction of the cytosolic tissue transglutaminase may be involved in the TGF-beta 1-induced pathways of apoptotic cell death in McA-RH7777 hepatoma cells.     

Expression of the cell cycle inhibitor p27KIP1 is a new prognostic marker associated with survival in epithelial ovarian tumors

This case-control study was designed to identify factors associated with long-term survival. We examined two groups of patients with epithelial ovarian cancer, one group of long-term survivors (> 5 years) and one group of short-term survivors (< 2 years), for levels of expression of p53 and p27KIP1 proteins (as both proteins have been shown to be independent prognostic markers in tumors other than ovary) and the relationship with patient survival. Our findings show that p27KIP1 expression, in contrast to p53 expression, is positively associated with long-term survival in univariate analysis (P = 0.001), in analyses stratified by residual disease (P = 0.02) or performance status (P = 0.02), the two strongest prognostic factors for ovarian cancer, as well as multivariate analysis (P = 0.002) adjusting simultaneously for age, tumor stage, residual disease, performance status, and grade of differentiation. Therefore, immunostaining for levels of p27KIP1 expression may have potential as a new prognostic factor in the management of ovarian cancer.     

Erythroid potentiating activity of tissue inhibitor of metalloproteinases on the differentiation of erythropoietin-responsive mouse erythroleukemia cell line, ELM-I-1-3, is closely related to its cell growth potentiating activity

The erythroid-potentiating activity (EPA) of the tissue inhibitor of metalloproteinase-1 (TIMP-1) was re-examined using ELM-I-1-3, a mouse erythroleukemia cell line, which responded well to erythropoietin. Depletion of pre-existing TIMP-1 from fetal calf serum in culture medium using monoclonal antibody suppressed erythropoietin-induced differentiation as measured by the induction of hemoglobin, commitment assay and globin mRNAs. The removal of TIMP-1 also suppressed the proliferation of ELM-I-1-3 as measured by cell number and de novo DNA synthesis. These changes were reversed by the addition of purified TIMP-1 to the culture medium. Anti-TIMP-1 antibody also blocked both hexamethylene bisacetamide (HMBA)-induced erythroid differentiation and the proliferation of both ELM-I-1-3 and Friend erythroleukemia cells. Considering previous reports analyzing the chemical induction of Friend mouse erythroleukemia cell differentiation, our results suggest that erythropoietin- or HMBA-induced erythroid differentiation might also be coupled with cell proliferation. Our 3H thymidine-uptake experiment shows that TIMP-1 removal was also effective in the inhibition of cell growth of various other cell lines in addition to erythroleukemia cell lines. These results suggest that EPA action of TIMP-1 on erythroid leukemia cell lines is closely related to its activity to promote the cell growth of various cell lines and cells including erythroleukemia cell lines.     

Transforming growth factor-beta (TGF-beta)-mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-beta and a progressive increase in TGF-beta susceptibility of anti-tumor CD4+ T cell function

The present study deals with the effect of transforming growth factor-beta (TGF-beta) on anti-tumor immune responsiveness at various stages of the tumor-bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1-3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and macrophage-activating factor (MAF)/interferon-gamma (IFN-gamma) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4+ T cells and antigen-presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The IL-2-producing capacity of CD4+ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor-bearing stages. In contrast, the capacity of CD4+ T cells to produce MAF/IFN-gamma was not affected but was maintained at high levels even late in the tumor-bearing state. The addition of recombinant TGF-beta (rTGF-beta) to cultures of spleen cells from various tumor-bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF-beta-induced suppression varied depending on which tumor-bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1-3 wk) tumor-bearing stages but increased in cells from donor mice at later tumor-bearing stages. Thus, spleen cells from late tumor-bearing stages with weak but significant IL-2-producing and considerable MAF/IFN-gamma producing capacities failed to produce these lymphokines when rTGF-beta was present in cultures. A progressive increase in the TGF-beta susceptibility was also observed for IL-4-producing Th2 as well as IL-2/MAF-producing Th1 cells. In addition, increased levels of TGF-beta were detected in plasma from tumor-bearing mice at late stages. Taken together, these results indicate that tumor-bearing mice exhibit enhanced production of TGF-beta as well as a progressive increase in the susceptibility of anti-tumor CD4+ T cells to TGF-beta-induced suppressive mechanisms.     

A case of lymphoid interstitial pneumonia in a 3-month-old boy not associated with HIV infection: immunohistochemistry of lung biopsy specimens and serum transforming growth factor-beta 1 assay

Women have a higher stress fracture rate than men in military studies, although the exact cause of this is not clear. Hyperpronation has been implicated as a potential risk factor for injury. In this prospective observational study, we measured subtalar joint range of motion in 101 women (ages 20-27 years) enrolled in Marine Corps Officer Candidate School in June 1994. The purpose of this study was to identify risk factors for injury in female Marine Corps officer candidates. The primary area of interest was the association between the amount of subtalar joint range of motion and stress reactions. Questionnaires were administered that explored previous physical activities, sports participation, and menstrual history. Anthropometric measurements were performed, including subtalar joint range of motion. During the 10 weeks of physical training, 11.5% of the women (N = 12) had stress reactions compared with 7% of the men (N = 10). There was no statistically significant difference in the means of subtalar joint range of motion in the stress reaction group compared with the non-stress reaction group. Differences in stress reaction rate across quartiles of subtalar joint range of motion were not significant. Those women who ran fewer miles (< or = 2.8 miles per session) before training had a higher rate of stress reactions (p < 0.04). Younger individuals (< 23 years) had a higher rate of stress reactions (p < 0.01). Women with fewer menstrual periods (< 10 per year) had a higher rate of stress reactions (p < 0.02). A narrow pelvis (< or = 26 cm) was associated with a higher rate of stress reactions (p < 0.09). We conclude that an increased subtalar joint range of motion is not a risk factor for stress reactions in women. However, further studies with a larger study population should be performed to confirm these findings.     

p27Kip1: a key mediator of retinoic acid induced growth arrest in the SMS-KCNR human neuroblastoma cell line

Retinoic acid (RA) treatment of SMS-KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal differentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR while p15INK4B and p16INK4A were not detected. RA induced an increase in the expression of p27Kip1 but not p21Cip1. Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27Kip1. These results indicate that changes in the level of p27Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.     

Correlative alteration of thromboxane A2 with antigen-induced bronchoconstriction and the role of platelets as a source of TXA2 synthesis in guinea pigs: effect of DP-1904, an inhibitor of thromboxane synthetase

Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.     

The growth inhibitory effect of N1,N12-bis(ethyl)spermine in small cell lung cancer cells is maintained in cells expressing the c-myc and Ha-ras oncogenes

OBJECTIVE: The purpose of this study was to assess the diagnostic role of MR arthrography in patients with tendinopathy or rupture of the long biceps tendon. MATERIALS AND METHODS: MR arthrograms of 42 consecutive patients with arthroscopic or surgical confirmation of diagnosis (16 normal biceps tendons, 19 with tendinopathy, and seven with ruptures) were analyzed independently by two radiologists. Visibility of the biceps tendon, caliber changes, contour irregularities, and signal intensities were assessed separately in the parasagittal and axial planes. In addition, the two radiologists made an overall evaluation of abnormalities of the biceps tendon using both MR imaging planes. RESULTS: The most reliable MR findings for tendinopathy were caliber changes (sensitivity was 59% for observer 1 and 82% for observer 2; specificity was 88% and 64%, respectively) and signal abnormalities (sensitivity, 77% and 88%, respectively; specificity, 75% and 43%, respectively) in the parasagittal plane. Absence of visualization of the tendon in the parasagittal plane was a reliable sign for rupture (sensitivity, 86% and 86%, respectively; specificity, 94% and 87%, respectively). The overall sensitivity for detecting abnormalities (tendinopathy or rupture) was 92% for observer 1 and 89% for observer 2. Specificity was 56% and 81%, respectively. CONCLUSION: MR findings of tendinopathy and rupture of the biceps tendon are subtle. However, the combination of several MR criteria in two imaging planes makes a reasonably accurate diagnosis possible. The biceps tendon should not only be assessed in the bicipital sulcus on axial images but also on parasagittal images.