Fig. 1.
Molecular characterization of N1IC-induced mammary tumors. (A) Northern blot analysis of total cell RNA from WT mammary glands of virgin (V), pregnant (P), lactating (L), and postinvolutional (PI) female mice and from regressing (R) and nonregressing (NR) N1IC-induced mammary tumors (two specimens of each class). The same membrane was sequentially hybridized (without stripping), with specific probes detecting the indicated RNA species. The relative Myc levels in the controls were approximately V 1.0, P 2.0, L 1.0, and PI 1.5. There was an ≈7-fold increase in Myc expression in regressing tumors relative to the amount in normal lactating glands. A similar Myc increase (≈7.5-fold on average) was detected in nonregressing carcinomas compared with corresponding control postinvolutional glands. Expression of the transgenic N1IC resulted in up-regulation of the endogenous Notch1 mRNA (row mN1, lanes 5–8). The level of mN1 transcripts in the control specimens was below detection limits by Northern blot analysis under our conditions. Hybridization to 18S rRNA (loading control) was performed to normalize the data for quantitation by using a PhosphorImager (Molecular Dynamics). (B) Northern blot analysis shows that the level of Myc mRNA expression in MMTV-Myc-induced carcinomas (≈45-fold greater than normal) is approximately six times higher than that found in nonregressing N1IC tumors. The transcript derived from the MMTV-Myc transgenic construct (14) is longer than the endogenous Myc mRNA. Lanes 1 and 2 are the same as Myc lanes 7 and 8 in A (different exposure times). (C) Southern blot analysis of EcoRI-digested DNA from a lactating mammary gland of a female mouse with an MMTV-N1IC/Mycfl/fl/Wapcre genotype after a second pregnancy, to assess the level of Cre-mediated DNA excision (lane 2) from the “floxed” (fl) Myc locus (generation of Δ allele). The control DNA (floxed allele; lane 1) was prepared from a nonregressing tumor of an animal with the same genotype. (D) Comparative Northern blot analysis of Myc mRNA expression levels at 2 weeks postpartum between N1IC-induced regressing tumors developed in females carrying MMTV-N1IC in functional Myc background (R; lanes 1 and 2; same as Myc lanes 5 and 6 in A) and tumor-free lactating glands of MMTV-N1IC/Mycfl/fl/Wapcre mice, in which Cre-mediated recombination (r) at the Myc locus had occurred. RNA was extracted from glands after the first pregnancy (lanes 3 and 4) or after a second pregnancy (lane 5).