WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

Abstract

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline‐regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1‐mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1‐target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC‐rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci

“…In two independent clones (see Materials and methods), termed UTA-L1 and UTA-L2 (`L' for LAZ3, the former name of BCL6), the basal (in presence of tetracycline) expression of BCL6fg is very similar to BCL6 endogenous levels while removal of tetracycline leads to an induction of BCL6fg mRNA that is detectable after a few hours (data not shown) and reach several hundred-fold after 48 h (Figure 1a). This is similar to other inducible clones derived from the UTA-6 parental cells (Englert et al, 1995). The expression of the BCL6fg protein at the expected size was veri®ed by Western blot while immuno¯uorescence experiments gave a nuclear punctated or speckled pattern very encompassing the 3' coding and non-coding human BCL6 cDNA (see Materials and methods).…”

“…Note that the apoptosis is not synchronous in that few apoptotic cells can be found by DAPI staining after 2 ± 3 days of BCL6fg expression (data not shown) while a more prolonged expression (5 ± 6 days) is needed to induce the death of almost the entire culture (Figures 3b and 4b). In this respect, BCL6-mediated apoptosis resembles that observed upon overexpression of WT-1, another kruÈ ppel-like transcriptional repressor (Englert et al, 1995). In sharp contrast, despite the negative e ect of Znfg on cell growth, UTA-Z1 and UTA-Z2 cells do not appear to undergo apoptosis by any of the criteria described above (Figures 3b, 4b, 5a and b).…”

“…UTA-6 cells are stably transfected with the tTA chimeric activator which comprises the activation domain of the herpes virus VP16 fused to the E. coli tetracycline repressor. tTA binds the TET operator upon withdrawal of tetracycline from the culture medium (Englert et al, 1995). The parental UTA-6 cells which express a very low level of endogenous BCL6 mRNA (data not shown) were stably transfected with a construct placing a¯agged version of the human BCL6 cDNA (thereafter referred to as BCL6fg, Dhordain et al, 1995) under the control of the TET operator.…”

“…The U2OS-derived parental UTA-6 clone has been previously described (Englert et al, 1995) and was routinely maintained in Dulbecco medium supplemented with 10% fetal calf serum (Boehringer) (thereafter referred to as DF10), G418 (GIBCO ± BRL) 500 mg/ml and tetracycline (Sigma) 2 mg/ ml. It constitutively expresses the TET-VP16 inducible transactivator (Gossen and Bujard, 1992).…”

Nuclear targeting of Bax during apoptosis in human colorectal cancer cells

“…Furthermore, growth factors may also contribute towards cell survival since growth factor withdrawal can lead to apoptosis (Oltvai et al, 1993;White 1996). There are numerous examples demonstrating that EGF can prevent apoptosis in response to a wide variety of stimuli (Coles et al, 1993;Luciano et al, 1994;Nakajima et al, 1994;Fabregat et al, 1996;Garcia-Lloret et al, 1996), and inhibition of EGFR expression can induce apoptosis (Englert et al, 1995). Since activation of EGFRs may be involved in stimulating colonic epithelium proliferation and in antagonizing apoptosis, one potential mechanism to suppress colorectal cancer may include modulation of apoptotic pathway(s) by inhibiting EGFR using anti-EGFR mAbs.…”