Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression - PubMed (original) (raw)

H Chen et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

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Figures

Figure 5

Figure 5

(A) Oligonucleotide sequences used for gel mobility-shift analysis: WT, derived from the hASH1 promoter (base pairs −265 to −247) containing the class C site (underlined); mutant, identical to WT except for five point mutations in the class C site (underlined); CT7–8, an irrelevant probe derived from the calcitonin gene promoter (38); and 2NB, containing the two N-box HES-1 binding sites (underlined) in the rat HES-1 promoter (27). (B) Competitor studies of the hASH1 promoter class C site using NCI-H157 nuclear protein extracts (4 μg), WT probe, and competitors shown in A. Lanes: 1, free probe; 2, no competitor; 3 and 4, 50- and 150-fold excess WT; 5 and 6, mutant; 7 and 8, CT7–8; 9 and 10, 2NB.

Figure 1

Figure 1

(A) Western blot analysis of HES-1 protein in lung cancer cell lines, grouped by hASH1 mRNA expression status. Extracts from 1 × 106 cells separated by SDS/PAGE were analyzed using polyclonal rabbit antisera raised against a C-terminal peptide common to rat and human HES-1. (B) Lysates (5 μl of 50 μl) from in vitro translation reactions containing no plasmid (control) or a HES-1 expression construct, as well as an extract from NCI-H157 cells, were analyzed for HES-1 protein by Western blotting as above.

Figure 2

Figure 2

Induction of HES-1 in SCLC cells. (A) DMS53 cells containing a tetracycline-inducible transcriptional activator were stably transfected with HES-1 plasmids driven by a tetracycline response element, treated with carrier or doxycycline (1 μg/ml) for various time periods, and analyzed for HES-1 protein expression by Western blotting as described in Fig. 1. Lanes: 1, carrier-treated DMS53-HES-1 (sense orientation HES-1 construct) cells, 48 hr; 2, doxycycline-induced DMS53-HES-1 cells, 48 hr; 3, carrier-treated DMS53-HES-1 cells, 146 hr; 4, doxycycline-treated DMS53-HES-1 cells, 146 hr; 5, carrier-treated DMS53-vector cells, 48 hr; 6, doxycycline-treated DMS53-vector cells, 48 hr; 7, carrier-treated DMS53-rev-HES-1 (antisense orientation HES-1 construct) cells, 48 hr; 8, doxycycline-treated DMS53-rev-HES-1 cells, 48 hr. (B) Effect of HES-1 induction on hASH1 mRNA levels. Total RNA (20 μg) from DMS53 cells transfected with tetracycline-inducible constructs, and treated with carrier (TET−) or 1 μg/ml doxycycline (TET+) for 8, 48, and 146 hr, was analyzed for hASH1 and GAPDH mRNA as described in Materials and Methods. In the bottom panels, the ratios for the relative intensities of the hASH1 and GAPDH hybridization signals were calculated from PhosphorImager values.

Figure 3

Figure 3

(A) Schematic representation of the class C site (solid box, bp −258) in the proximal hASH1 repressor region (open box). The striped box indicates the basal hASH1 enhancer (base pairs −234 to −46). (B and C) Transient transfection assays of hASH1 promoter constructs. Fragments of the hASH1 promoter region containing 5′ flanking sequence (shaded box), the candidate class C site (open box labeled with the letter C), the generalized enhancer region (striped box), the transcription initiation site (at position +1), and a short 5′ untranslated region were subcloned into the luciferase reporter vector pGL2 and then cotransfected with CMV–β-galactosidase and either HES-1 (shaded bars) or control (solid bars) vectors into DMS53 cells [HES-1(−), hASH1(+)] (B) and NCI-H157 cells [HES-1(+), hASH1(−)] (C). Luciferase activity is normalized to CMV–β-galactosidase and expressed in relation to the most active construct (plasmid −234/+37). The letter X over the class C site represents mutation of this motif.

Figure 4

Figure 4

Gel mobility-shift analysis of the hASH1 promoter class C site. (A) Cell-type-specific shift patterns. Nuclear protein extracts (4 μg) from native cells (lanes 2–5) or from DMS53 cells expressing HES-1 (lane 9) or control constructs (lanes 6–8) were incubated with a WT probe, containing the class C site from the hASH1 promoter. (B) Effect of HES-1 antisera on complexes with the hASH1 promoter class C site. NCI-H157 nuclear extracts (4 μg) and various antisera were preincubated for 30 min, then incubated with WT probe. Lanes: 1, free probe; 2, extract with buffer alone, no antisera; 3, preimmune sera; 4, HES-1 antisera; 5, control hASH1 antisera.

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