Abstract
The role of transforming growth factor (TGF) beta type II receptor in reversing the malignant phenotype of human breast cancer MCF-7 cells was examined. MCF-7 cells were insensitive to TGF beta 1 and expressed undetectable levels of cell surface TGF beta type I receptor (RI) and type II receptor (RII) by cross-linking with 125I-TGF beta 1. Stable transfection of a RII expression vector yielded 3 transfectants with varying levels of exogenous RII mRNA and protein levels. Expression of RII also increased TGF beta 1 binding to RI in all 3 clones. Proliferation of RII-positive clones was inhibited by exogenous TGF beta 1 in a dose-dependent manner, whereas the control clones remained TGF beta-insensitive. The RII transfectants were growth arrested in monolayer culture at saturation densities which were 41-66% of that of the Neo controls. They also showed reduced clonogenicity in soft-agarose. Tumorigenicity in ovariectomized, estrogen-supplemented nude mice was delayed in transfectants with low RII levels. Transfectants expressing high levels of RII showed a large reduction in tumorigenicity as well as a longer delay in tumor formation. Tumor growth was associated with loss of exogenous RII expression in transfectants. The results indicate that when systems for TGF beta signal transduction are intact, reconstitution of the TGF beta receptor system can lead to reversion of malignancy in cells lacking RII.
Highlights
The role of transforming growth factor (TGPFt)ype I1 nique to suppress autocrine TGFp activity in two colon carcireceptor in reversing the malignant phenotype of hu- noma cell lines (Wu et al, 1992, 1993).Repression of endogemanbreastcancerMCF-7 cells was examined
RNAAnalysis-TotalRNAfromMCF-7 cells in culture and xenografts from tumor-bearing nude mice was extracted by guanidine thiocyanate homogenization and ultracentrifugation through a cesium gradient essentially as described by Chirgwin et al (1979).In order to measure receptor type I (RI) and RII mRNA levels, we constructed RI and RII riboprobe
A fragment of human RI cDNA(588 bp)was obtained by polymerase chain reaction using the sense primer 5"GACCAGTGTGCTTCGTCTGC-3' and the antisense primer 5'-GCTGGC"TCCTTGGGTACC-3' according tothe sequence published by Franzen et al (1993)
Summary
Receptor Cross-linking-Human TGFp, was purchased from R & D Systems (Minneapolis,MN) and iodinated by the chloramine T method. Cells (x2IO3or 6 x lo3)were ple cloning site contains a minimal human cytomegalovirus promoter suspended in 1 mlof 0.4% sea plaque-agarose in McCoy's medium with heptamerized tet-operators. This promoter has no activity without containing 10% FBS and plated on top of a 1-ml underlayer of0.8%. This plasmid (1 pg) and the RII-containing had been ovariectomized and 1.7 mg of 17p-estradiol 60-day extended plasmid (IO pg) werelinearized and transfected into one of the MCF-7 release pellets (Innovative Research of America, Toledo, OH)were limiting dilution clones designated MCF2O with a Bio-Rad electropora- placed subcutaneously as described by Gottardis et al (1988) 1 week tor at 250 V and 960 microfarads.
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