p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development

Abstract

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.

Figure 1.

Morphogenesis of WT and p19^ARF null mammary glands during development. Parts of whole mounts of WT and p19^ARF null mammary glands showing the morphological differences and similarity in both groups during development. (A) Various time points of virgin mammary gland during development and (B) stages of mammary gland development at indicated stage-points.

Figure 2.

Rate of cell proliferation and apoptosis in WT and p19^ARF null mammary glands during development. (A) Rate of cell proliferation presented as mean percent of BrdU LI (±SD) in a bar graph calculated from 4–6 mammary gland/stage-point in each group and statistically analyzed using the two-sided Student's t test. On the top of the bar graph, examples of mammary gland sections showing the proportion of number of cells stained positively for BrdU in WT vs. p19^ARF null at 8 days of involution. (B) Bar graph showing mean percentage apoptotic cells (±SD) in WT and p19^ARF null mammary glands at indicated time points during involution. On the top, examples of mammary gland sections showing the proportion of number of positive apoptotic cells stained in WT vs. p19^ARF at 8 days of involution (p < 0.02).

Figure 3.

p19^ARF expression level during mammary gland development. Mammary glands from WT and p19^ARF null mice of 129Svj × C57BL/6 background were analyzed. (A) Protein levels. Equal amounts of protein extracts (500 μg/lane) of WT mammary glands at indicated stage-point during development were analyzed by Western blot for p19^ARF and actin levels using rabbit polyclonal antibody specific for mouse p19^ARF or actin mAb. Protein bands were detected by ECL detection mixture, scanned, quantified, normalized to the levels of actin, and presented in bar graph. The positive control is BALB/c3T3 10(1) cells. (B) Expression level of p19^ARF and β-actin analyzed by RT-PCR on mammary glands of WT and p19^ARF null mammary glands at indicated stage-points. Positive control is a PCR product of tail DNA of WT mice.

Figure 4.

Upregulation of p19^ARF and cellular localization in hormonally stimulated normal mammary gland. (A) Western blot analysis of p19^ARF protein levels in hormonally stimulated normal mammary glands of virgin WT mice at indicated time points after receiving a pituitary isograft. The positive control and negative controls are protein extract from BALB/c3T3 10(1) (ARF-wild-type, p53-null) cells and 3T3 cells of WT p53, respectively. Below, parts of whole mounts showing changes in morphogenesis at indicated time points. Magnification, ×5. (B) Western blot analysis of p19^ARF and actin protein level in mammary glands of mice hormonally stimulated by different hormones as indicated. Also, parts of whole mounts of mammary glands showing change in morphogenesis at individual or combination of hormone stimulation compared with untreated mammary gland (control). (C) Localization of p19^ARF in mammary glands at different hormonal conditions. p19^ARF stained in red showing overexpression predominantly in luminal epithelial cells in mammary glands stimulated with progesterone, and E+P, whereas weak staining or in PRL or E alone. Two negative controls were included; untreated mammary gland of WT virgin mouse (C) and p19^ARF null mammary gland of pregnant mouse. K14, in green, is specifically myoepithelial. Images were taken at (40×) magnification using confocal microscopy.

Figure 5.

Immortalization of p19^ARF null mammary epithelial cells in vivo. (A) A graph presenting percent of area filling the fat pad in 5–6 animals per group for seven transplant generations. The percent of the area filling the fat pad was calculated by measuring the length and width of the ductal area filling the fat pad proportional to the total area of the fat pad using microscope scale. (B) Representative histology sections of WT and p19^ARF null mammary outgrowths after six transplants showing BrdU incorporation detected by immunoflorescent staining. A bar graph presenting BrdU labeling index (LI) statistically analyzed from four outgrowths using two-sided Student's t test. (C) Whole mounts of the 6th transplant of WT and p19^ARF null mammary outgrowths showing the difference in the area filled the fat pad in each cell type. Also, histology sections of the 6th transplant of WT and p19^ARF null outgrowths stained for p21^Cip1, p27^Kip1, p16^INK4a, p53, or no primary antibodies (as negative control). Notice the lack of p21^Cip1-positive cells in the p19^ARF null cells compared with WT in enlarged part of each section.

Figure 6.

Histology sections of WT and p19^ARF null mammary glands during involution stained for p21^Cip1. A representative sections of WT and p19^ARF null mammary glands at 2, 8, and 15 days of involution stained immunohistochemically for p21^Cip1 using antibodies specific for mouse p21^Cip1 (PharMingin Laboratories). Arrows pointed at nuclear and cytoplasmic staining at 2 and 8 days of involution, respectively in the WT, and few nuclear positive cells at 8 days of involution in p19^ARF null. Magnification, ×40.