FIGURE 1

Np73 inhibits multiple cellular differentiation processes. (a) The transactivation function of the three p53 family members (p53, TAp63, TAp73) is inhibited by coexpression of Np73 isoforms generated by both alternative splicing of exon 2 (N^AS) or alternative promoter usage (N^AP). In detail, H1299 cells were cotransfected with 200 ng luciferase reporter plasmid containing p53 consensus binding sites,²⁵ 50 ng p53, TAp63 or TAp73 expression plasmid²⁶ and 400 ng of the indicated Np73 plasmid.²⁷ Luciferase activity was determined 48 h after transfection. (b) C2C12 cells were induced to differentiate into myotubes by incubation in medium containing 2% horse serum. RNA was isolated from the cells at the indicated time points (h) and expression of p53, TAp63 and TAp73 was measured by semiquantitative RT-PCR. Expression of the housekeeping gene GAPDH is shown as a control. (c) C2C12 cells were transduced with retrovirus generated by transient transfection of the amphotropic packaging cell line PT67 (Becton Dickinson) with either empty vector (mock) or the pQCXIP-Np73 vector. Transduced cells were selected in growth medium containing 1.5 g/ml puromycin. Cells were either grown in growth medium or differentiated for 5 days with 2% horse serum. Differentiation into multinuclear myotubes was analyzed by immunofluorescence staining for myosin heavy chain (MF 20 antibody, Developmental Studies Hybridoma Bank). Nuclei were counterstained with DAPI. (d) C2C12 myoblasts were stably transduced with retroviral vectors (mock, Np73 or dominant-negative p53 p53DD) and analyzed for myosin heavy chain (myHC) expression by Western blot before and after induction of differentiation. (e) C2C12 myoblasts (mock or Np73) were differentiated for 5 days in medium containing 5% FCS in the absence or presence of 300 ng/ml BMP2 (kindly provided by Walter Sebald, University of Würzburg, Germany) and stained for the osteoblast marker gene alkaline phosphatase as described.²¹ (f) C2C12 myoblasts treated as in (e) were analyzed by semiquantitative RT-PCR for expression of muscle (myHC) or osteoblast (alkaline phosphatase, AP; osteocalcin) marker genes. (g and h) SH-SY5Y neuroblastoma cells were transfected with pQCXIP (mock) or pQCXIP-Np73 plasmids and selected in 1 g/ml puromycin. Cells were induced to differentiate in 1 M all-trans retinoic acid (ATRA), analyzed for (g) expression of the neuronal marker gene neurofilament by semiquantitative RT-PCR and (h) neurite extension by phase contrast microscopy. Sequences of primers used for RT-PCR are available upon request. (i) Diagram of the conditional Np73 transgene construct. In the uninduced state the broadly active -actin promoter drives expression of a GFP-stop cassette flanked by loxP sites. Recombination induced by Cre results in excision of the GFP-stop cassette and places the Np73 cDNA (Np73 isoform generated by alternative splicing of exon 2) under the control of the -actin promoter. Expression of Np73 is coupled to a human placenta-like alkaline phosphatase cDNA via an IRES sequence. Binding sites for primers used in subsequent PCR and RT-PCR analyses are indicated. (j) H1299 cells were transfected with the transgene construct by electroporation and infected with 50 moi of Cre-expressing adenovirus (+Cre; kindly provided by Anton Berns) 24 h later to induce recombination. Expression of Np73 and -actin was assessed by Western blot (anti-p73 ER15, Merck Biosciences; anti-actin, Abcam). (k) The GFP-stop cassette in the inactive Np73 transgene plasmid (N^flox) was excised with recombinant Cre enzyme (Becton Dickinson) in vitro resulting in the active N^Cre plasmid. Inhibition of the transactivation function of the three p53 family members (p53, TAp63, TAp73) was analyzed by coexpression of either N^flox or N^Cre as outlined in Figure 1a. Reporter activity in the presence of p53, TAp63 and TAp73 alone was set as 100. The actual transactivation potential is shown in Figure 1a. (l) Recombination of the Np73 ^flox transgene can be induced in vitro by expression of Cre. Dermal fibroblasts were isolated from Np73 ^flox transgenic mice generated by pronuclear injection of the Np73 ^flox transgene construct into mouse embryos. Recombination was induced by infection with 100 moi of Adeno-Cre. Left panel: recombination efficiency was analyzed by PCR using primers 1/5. 1, no template control (NTC); 2, uninfected; 3, Cre-infected. IL2 was amplified as a control. Right panel: Expression of GFP (primers 2/3) or Np73 (primers 4/5) was analyzed by semiquantitative RT-PCR on RNA isolated from mock- or Cre-infected transgenic fibroblasts. Controls without reverse transcriptase (-RT) are indicated. (m) Muscle satellite cells were isolated from Np73 ^flox transgenic mice as previously described¹⁶ and cultured in Ham's F10 medium supplemented with 20% FCS and 2 ng/ml bFGF (Invitrogen). Cells were infected with 100 moi Adeno-Cre or Adeno-GFP as a control and either incubated in growth medium (GM) or induced to differentiate for 30 h in differentiation medium (DM) containing 2% horse serum (left panel: phase-contrast microscopic images; right panel: semiquantitative RT-PCR analysis of Np73, ₁-actin, myosin heavy chain (myHC) and GAPDH expression). (n and o) Recombination of the Np73 ^flox transgene can be induced in vivo. Np73 ^flox transgenic mice were crossed to Mx1-Cre mice.²³ Cre expression in Np73 ^flox/Mx1-Cre double transgenic offspring mice was left uninduced (–) or induced (+) by three intraperitoneal injection of pI-pC (250 g) at 2-day intervals. At 5 days after the last injection (n) DNA and (o) RNA was isolated from various tissues and analyzed for recombination efficiency by PCR with primers 1/5 and for Np73 expression by RT-PCR with primers 4/5. Ta, tail; Sk, skin; Mu, muscle; Te, testis; In, intestine; Br, brain; Li, liver; Lu, lung; Sp, spleen; Th, thymus; NTC, no template control. (p) Female Np73 ^flox transgenic mice were crossed to male CMV-Cre mice (X^Cre/Y). DNA from offspring mice was analyzed for the Np73 transgene by PCR with primers 4/5, for the Y-chromosome (YMT) and for IL2 as a control. The genotypes for 30 newborn mice are given in the table. Results of equivalent crosses of male CMV-Cre mice with females of a different conditional transgenic line are included as a control