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. 1999 Aug 17;96(17):9827-32.
doi: 10.1073/pnas.96.17.9827.

Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion

M R Young  1 , J J Li, M Rincón, R A Flavell, B K Sathyanarayana, R Hunziker, N Colburn
Affiliations

Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion

M R Young et al. Proc Natl Acad Sci U S A. .

Abstract

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis in vivo has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant c-jun (TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14-TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-O-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.

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Figures

Figure 1
Figure 1
Expression of K14-TAM67 in transgenic mice. (A) Expression of TAM67 was determined by RT-PCR analysis of RNA isolated from skin. Founders 1, 2, and 3 (lanes 1–3), negative littermate (lane 4). RNA indicates the TAM67 PCR product of the cDNA generated by RT. DNA indicates the DNA product from PCR of the genomic DNA contaminating the RNA samples. β-actin is the RT-PCR product from the mouse β-actin gene. (B) TAM is expressed in epidermis, tongue, and cervix, but not dermis, liver, or kidney. RNA was subjected to RT-PCR using primers to amplify mouse K14 (mK14), TAM-hGH, β-actin mRNA, or 18s RNA.
Figure 2
Figure 2
TAM67 blocks TPA-induced AP-1 activation. The ears from two B6D2/N1/AP-1Luc/TAM67 mice and two TAM67-negative siblings were treated for 16 hr with acetone (right ear) or with 10 nmol of TPA (left ear). Tissue was isolated and lysed overnight, and luciferase activity was determined. The mean range for samples from two mice per group are shown. Similar results were seen on repetition of this experiment.
Figure 3
Figure 3
TPA induces ODC, plasminogen activator inhibitor-2, and MMPs equally in transgenic and control mice. (A) Epidermal RNA from four control or four B6D2/N1/TAM67 transgenic mice, exposed to acetone or TPA for 6 hr, was used as a template to generate a 32P-labeled cDNA probe for expression array analysis (CLONTECH). RT probes were hybridized to individual cDNA blots overnight and washed, and gene expression was determined by scanning with a Molecular Dynamics PhosphorImager. Expression levels were determined densitometrically and were standardized to two or more of the housekeeping genes. Two representative sections from the arrays are shown. (B) Epidermal RNA from treated skin was reverse-transcribed, and 10%, 20%, or 40% of the cDNA product was amplified by 20 cycles of PCR in the presence of [33P]dCTP. The PCR products were separated by PAGE. Gene expression was determined by scanning with a Molecular Dynamics PhosphorImager. PCR amplification was determined to be linear when 2-fold additions of cDNA resulted in 2-fold amplification of product. Results are shown for four independent mice: two control, two transgenic. Col-1, collagenase-1; ST-3, stromelysin-3.
Figure 4
Figure 4
TPA-induced hyperproliferation is not inhibited by TAM67. (A) Sections from dorsal skin of B6D2/N1/TAM67 (Right) and negative (Left) siblings treated with a single dose (Upper) or four twice-weekly doses (Lower) of TPA and stained with H&E 24 hr (Lower) or 48 hr (Upper) later. (B) Sections from dorsal skin of control mice (lanes 1–4) and TAM67 mice (lanes 5–8) were treated with a single dose of acetone (lanes 1 and 5) or TPA and 24 hr (lanes 2 and 6) or 48 hr (lanes 3 and 7) later stained with H&E, or treated with four twice-weekly doses of TPA (lanes 4 and 8) and 24 hr later stained with H&E. C57BL/6 (lane 9) was treated with four twice-weekly doses of TPA and stained 24 hr later. Sections were visualized under laser-induced confocal microscopy. The thickness of the epidermis was measured as described in Materials and Methods. An average of four measurements from two images from each section from two different animals (n = 16) for each time point was made.
Figure 5
Figure 5
Papillomagenesis is markedly inhibited by TAM67. (B6D2F1xDBA/2)N2.5 TAM67 (generated by crossing N3 males with N2 females), their negative siblings, and B6D2/F1 control mice were treated with a single dose of DMBA (400 nmol) and followed by twice-weekly doses of TPA (10 nmol) as described in Materials and Methods. Development of papillomas was determined by palpation. N in Inset indicates the number of animals. A similar result was obtained with B6D2/N1/TAM67 transgenics.
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