Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Abstract

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185(neu). We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185(neu)-treated tumor cells but not in cells expressing internalization defective p185(neu) receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

Figure 1

Regions of homology of 3C to murine TBP1. Amino acids 1–41 of 3C correspond to amino acids 22–62 of mTBP1, and amino acids 42–73 of 3C correspond to amino acids 411–442 of mTBP1 (100% identical). The 3C C-terminal noncoding-region nucleotide sequence is 98% homologous to that of mTBP1.

Figure 2

Comparison and confirmation of the differentially expressed gene by Northern blot analysis and endogenous expression of TBP1 in U87MG vs. U87/T691. (A) B104-1-1 and NR6TintΔ cells were incubated with or without 7.16.4 (10 μg/ml) for 24 h before RNA isolation. Total RNA (10 μg) was loaded in each lane and was probed with human TBP1 cDNA probe. TBP1 expression was up-regulated with anti-p185^neu mAb (7.16.4) treatment in B104-1-1 cells (lanes 1 and 2) but was not changed in internalization defective NR6TintΔ cells (lanes 3 and 4). (B) The corresponding formaldehyde gel electrophoresis of total RNA is shown. (C) The U87/T691 subclone, an epidermal growth factor receptor (EGFR)-positive cell line phenotypically inhibited by the expression of a trans-inhibitory ectodomain form of p185^neu (T691 stop neu), showed increased endogenous levels of TBP1. Relative units are derived from scanning densitometry (Molecular Dynamics).

Figure 3

Confirmation of TBP1-transfected clones by RT-PCR analysis. We made first-strand cDNA from 3 μg of each total RNA by using the Superscript Preamplification System for first-strand cDNA Synthesis Kit (GIBCO/BRL). (A) Amplification of the transfected TBP1 cDNA by using the pBK-CMV vector-oriented T7 primer (5′-GTAATACGCTCACTATAGGGC-3′) and the TBP1 specifically designed primer C2 (5′-AGAAGAAAGCCAACCTAC-3′) shows 216-bp product bands in only the transfected clones and the pBK-CMV–TBP1 plasmid construct and not in parental cell lines of U87MG, SK-BR-3, and MCF-7. (B) Amplification of actin cDNA by using the rat β-actin control amplifier set (CLONTECH) shows a 764-bp amplified product from all the cell lines except the pBK-CMV–TBP1 plasmid construct. After RT-PCR, the products were run on 1.8% agarose gel to confirm the amplified product.

Figure 4

Reduction of cell proliferation by expression of human TBP1 in human cancer cell lines. Cell lines were plated in 96-well plates at 4,000 cells per well in 10% DMEM and allowed to attach overnight. MTT was given to the cells for 4 h. Cells were then lysed in 50% (vol/vol) SDS/20% (vol/vol) dimethyl sulfoxide and kept at 37°C overnight. Proliferation was assessed by reading OD at 570 nm by using an ELISA reader. The number of cells used in this assay was determined to be within the linear range for this cell type. (A) TBP1 cDNA-transfected SK-BR-3 subclones S2 and S6 had 50% and 57% proliferation inhibition, respectively, compared with parental cells. (B) TBP1 cDNA-transfected MCF-7 subclones M2 and M6 had 40% and 54% proliferation inhibition, respectively, compared with parental cells. (C) U87MG subclones expressing elevated TBP1, U1, and U17 had 34% and 38% inhibition of proliferation, respectively, compared with parental cells.

Figure 5

Inhibition of cell growth and transformation by human TBP1. (A) Anchorage-independent growth. Cells of each clone (n = 1,000) were suspended in a 1-ml top layer [0.18% agarose/10% (vol/vol) FBS/10% (vol/vol) DMEM] in 6-cm culture dishes containing a 3-ml cell-free feeder layer consisting of 0.25% agarose in DMEM supplemented with 10% FBS and 20 mM Hepes (pH 7.5). Colonies (>0.3 mm) were visualized and counted on day 28 for all cell lines after staining with p-iodonitrotetrazolium violet (1 mg/ml). Each cell line was examined in triplicate in three separate experiments. The numbers of colonies reported represent the mean of triplicate samples. (B) Tumor growth in athymic mice: comparison of parental U87MG cells and U87/TBP1 transfectants. Cells of each cell line (n = 1 × 10⁶) were injected subcutaneously on day 0, and tumor volume was recorded weekly. These data represent individual tumor growth curves for U87MG parental cells (■) and mean tumor volumes for the U87/TBP1 subclone (●). (U87MG, n = 7; U87/TBP1, n = 8.)