Mammary gland involution is a physiologic process which follows lactation and results in the rapid disappearance of the entire lobulo-alveolar compartment. Coincident with the onset of involution, milk protein gene expression ceases and alveolar cells undergo programmed cell death. We investigated whether p53 induces apoptosis during mammary gland involution in vivo and participates in tissue remodelling. Towards this end, we examined the process of involution in the presence and absence of functional p53 in mouse models; in wild-type mice, in transgenic mice which express SV40 T-antigen specifically in mammary tissue during pregnancy, and in mice which carry non-functional p53 alleles in their germline. Mammary gland whole mount and histological analyses revealed that involution and remodelling, with the concomitant disappearance of the lobuloalveolar structures, proceeded normally in the absence of functional p53. In addition, the absence of functional p53 did neither alter the involution related pattern of bax (death inducer) gene expression, nor the ratio of RNAs encoding bcl-xS (death inducer) to bcl-xL (survival inducer).
Li, M., Hu, J., Heermeier, K., Hennighausen, L. and Furth, P.A. (1996) Apoptosis and remodelling of mammary gland tissue during involution proceeds through p53 independent pathways. Cell Growth Diff. 7, 13-20.
Background
Mice which lack p53 are able to nurse their young suggesting that p53 is not required for
lactation. We approached the question whether p53 participates in the progression of
mammary gland involution in two mouse models. In a transgenic model in which an SV40
T-antigen (Tag) transgene is expressed in mammary alveolar cells during pregnancy and in
post-partum mice. This model enabled us to determine whether the sudden loss of functional
p53 during pregnancy in mammary epithelial cells would affect the initiation and
progression of apoptosis. Importantly, the rapid inactivation of p53 in mammary cells of
these mice left little opportunity for compensatory genetic modifications to occur. As a
second model we used mice which carry defective p53 alleles in their germ line. This model
enabled us to determine whether the loss of p53 in both mammary alveolar cells and in
additional cell types within the mammary fat pad interrupted the involutional process.
Involution in the presence and absence of functional p53 was assessed through the examination of lobulo-alveolar and cellular structures, and by monitoring the expression of genes linked to cell differentiation and PCD. Whole mount analyses enabled us to determine whether the absence of functional p53 led to a diminished loss of cells during involution. Histological sections were used to assess whether the collapse of lobulo-alveolar structures proceeded normally over the first three days of involution. The appearance of apoptotic cells during involution was monitored using in situ staining. Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess the expression of the apoptosis pathway genes, bax and bcl-x, and the milk protein genes, beta-casein, whey acidic protein (WAP) and alpha-lactalbumin.
Transgene
p53 null mice from the labs of Larry Donehower and Tyler Jacks were analyzed.
Mammary gland involution progresses normally in the absence of functional p53
Whole mount analyses were performed to determine if involution proceeded normally in the
absence of functional p53 as measured at the level of the lobulo-alveolar structure in the
whole organ. Mammary glands from transgenic WAP-Tag and control mice were examined at day
1 and day 13 of involution following the first pregnancy.
Apoptosis of mammary alveolar cells and collapse of lobulo-alveolar structures
progressed independently of functional p53
Histological analyses were conducted to identify mammary alveolar cells undergoing
apoptosis, and to determine whether the collapse of lobulo-alveolar structures was
affected by the absence of p53. In both Tag and control mice, numerous apoptotic cells
were present at day 1 of involution. At day 3 of involution the lobulo-alveolar structures
had collapsed in both Tag and control mice and equal numbers of apoptotic cells were
found.
Similar histological analyses were performed in p53 -/- and p53 +/+ mice which had completed three weeks of lactation before weaning and the induction of involution. Both lobulo-alveolar collapse and increased numbers of apoptotic cells were found in p53-/- and p53+/+ mice after three days of involution (image 4).
Expression of apoptosis genes
Mechanistic Implications
Keyword
apoptosis, cell cycle, p53
Submitted by: Priscilla A. Furth on April 30, 1996
Address
University of Maryland Medical School
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e-mail: furth@ncifcrf.gov