RII mRNA Levels Research Articles

Analysis of Transforming Growth Factor Receptor Type II in Patients with Agnogenic Myeloid Metaplasia (AMM).

We previously reported that blood TIMP-1 (Tissue Inhibitor of Metalloproteinase), OPG (Osteoprotegerin), and TPO (Thrombopoietin) levels are increased and blood MMP-3, and bone marrow SDF-1 levels are decreased in patients with AMM. Enhanced TGF-ß 1 activity with Transforming Growth Factor Receptor Type II (TGF ß RII) abnormalities is likely to be associated with these findings. It has been previously reported that TGF-ß RII levels are decreased in AMM patients. The current study was undertaken to explore the defects in the TGF-ß1 pathway in this disease. We cloned and sequenced the full length TGF ß RII cDNA from AMM patients and no mutations were detected on sequencing. We then determined the expression levels of TGF ß RII in the CD34+ progenitor cells by Real-Time RT-PCR and found it to be reduced in patients with AMM compared to normal volunteer CD34+ cells used as controls. Hence we cloned the promoter region of TGF ß RII (−1233 to +50) from the genomic DNA of Peripheral Blood Monocytes by PCR, using gene specific primers. On sequencing, no mutations were detected in the promoter regions. The same promoter region when cloned upstream of a luciferase gene, drove the transcription of the reporter gene similar to the levels observed with the promoter from controls. This augmented the absence of any structural abnormalities in the region of the TGF b RII promoter. We then analyzed the methylation status of TGF ß RII promoter using the technique, Sodium Bisulfite-PCR for Sequencing. Genomic DNA was modified using Sodium Bisulfite and the TGF ß RII promoter was amplified by PCR with Primers capable of amplifying both unmethylated and methylated DNA. The modified PCR products were cloned into TOPO TA sequencing vector from Invitrogen, CA and sequenced. The sequencing results revealed absence of any CpG hyper-methylation in the promoter region of TGF ß RII. To account for reduced TGF ß RII expression in the absence of coding region abnormalities, promoter abnormalities or hypermethylation, we determined Histone Deacetylase Activity HDAC activity in the CD34+ cells of AMM patients. There was an indirect correlation between HDAC activity and TGF ß RII mRNA levels implying HDAC mediated silencing of the TGF ß RII promoter in AMM patients.

The TGF-beta type II receptor in chronic myeloid leukemia: analysis of microsatellite regions and gene expression.

Genomic instability is one mechanism proposed to play a role in the disease progression of chronic myeloid leukemia (CML). Microsatellite regions in the type II transforming growth factor-beta receptor (TGF-beta RII) gene appear to be targets for mutation in some cancers displaying microsatellite instability (replication error phenotype, RER+). Furthermore, TGF-beta RII mutations in RER+ tumors have been associated with decreased TGF-beta RII mRNA levels. As TGF-beta is a potent negative growth regulator of hematopoietic cells, investigations were undertaken to determine whether inactivation of the receptor by microsatellite alteration might be involved in the progression of CML. Analysis of TGF-beta RII mRNA expression by RNase protection, with comparison of cells from the chronic, accelerated and blast phases of CML, showed no change in TGF-beta RII transcript levels during disease progression. However, during each phase of the disease, low levels of TGF-beta RII were detected when compared with the hematopoietic cells of normal donors. Furthermore, this decreased expression was also observed in the other myeloproliferative disorders, polycythemia rubra vera (PRV) and essential thrombocythemia (ET). The leukemia cell lines K562 and HL-60 had no detectable TGF-beta RII mRNA. Two microsatellite regions found altered in RER+ colon cancers were analyzed to establish if these sequences were aberrant in CML. No alteration was detected in either of these regions in any phase of the disease. These results suggest that alterations of the microsatellite regions in the TGF-beta RII gene are not involved in the progression of CML. Decreased expression of TGF-beta RII in CML cells and leukemia cell lines raises the possibility that altered expression of the receptor may play a role in the initiation and/or maintenance of the disease state.

Early castration‐induced upregulation of transforming growth factor β1 and its receptors is associated with tumor cell apoptosis and a major decline in serum prostate‐specific antigen in prostate cancer patients

BACKGROUND The mechanism behind castration-induced apoptosis in prostate cells is unknown, but data from other species suggest that transforming growth factor β1 (TGF-β1) may be involved. METHODS By using quantitative RT-PCR and immunohistochemistry, expression of TGF-β1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of core biopsies taken before and 2–11 days after castration therapy. The TGF-β responses were related to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA). RESULTS In normal prostate tissue, apoptosis was generally increased by castration, and apoptosis was accompanied by an increase in TGF-β1 and RII mRNA levels (P < 0.05). In tumors, apoptosis was seen only in 44% of the cases and in these, but not in the others, TGF-β1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing a prognostically favorable PSA response (nadir PSA <5 ng/ml), but not in the others, RI and RII mRNA levels were significantly upregulated (P < 0.05). CONCLUSIONS Short-term upregulation of TGF-β1 and its receptors is associated with apoptosis in human prostate and prostate cancer, and possibly with a favorable clinical outcome after castration therapy. Prostate 38:268–277, 1999. © 1999 Wiley-Liss, Inc.

Early castration-induced upregulation of transforming growth factor beta1 and its receptors is associated with tumor cell apoptosis and a major decline in serum prostate-specific antigen in prostate cancer patients.

The mechanism behind castration-induced apoptosis in prostate cells is unknown, but data from other species suggest that transforming growth factor beta1 (TGF-beta1) may be involved. By using quantitative RT-PCR and immunohistochemistry, expression of TGF-beta1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of core biopsies taken before and 2-11 days after castration therapy. The TGF-beta responses were related to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA). In normal prostate tissue, apoptosis was generally increased by castration, and apoptosis was accompanied by an increase in TGF-beta1 and RII mRNA levels (P < 0.05). In tumors, apoptosis was seen only in 44% of the cases and in these, but not in the others, TGF-beta1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing a prognostically favorable PSA response (nadir PSA <5 ng/ml), but not in the others, RI and RII mRNA levels were significantly upregulated (P < 0.05). Short-term upregulation of TGF-beta1 and its receptors is associated with apoptosis in human prostate and prostate cancer, and possibly with a favorable clinical outcome after castration therapy.

Decreased Stability of Transforming Growth Factor beta Type II Receptor mRNA in RER+ Human Colon Carcinoma Cells

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.

TNF-alpha regulates IL-4-induced Fc epsilon RII/CD23 gene expression and soluble Fc epsilon RII release by human monocytes.

We examined the regulatory effects of TNF-alpha on IL-4-induced gene expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) in human monocytes and IL-4-induced soluble Fc epsilon RII (sFc epsilon RII) release from monocytes. IL-4-induced Fc epsilon RII expression on the surface of monocytes was reduced by TNF-alpha as early as 1 day after culture and the effect of TNF-alpha increased with prolonged culture. The present analysis was designed to examine whether or not TNF-alpha could suppress IL-4-induced Fc epsilon RII mRNA expression and enhanced IL-4-induced sFc epsilon RII release. The addition of TNF-alpha to monocyte cultures with IL-4 significantly reduced Fc epsilon RII expression on the surface of monocytes and significantly increased sFc epsilon RII release from monocytes. Over time, there was an inverse relationship between the disappearance of cell surface Fc epsilon RII and the appearance of sFc epsilon RII in culture supernatants. Fc epsilon RII mRNA expression in monocytes cultured with IL-4 was not affected by TNF-alpha when examined at 6 h after cultivation. When the cells were cultured with TNF-alpha for more than 24 h, however, TNF-alpha down-regulated IL-4-induced Fc epsilon RII mRNA levels. This correlated with the kinetics of down-regulation of IL-4-induced Fc epsilon RII expression on the surface of monocytes by TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)