Apoptosis is a type of physiological cell death that occurs during development, normal tissue homeostasis, or as a result of different cellular insults. The phenotype of an apoptotic cell isrelativelyconsistentinmostcasesofapoptosisandinvolves at least changes in the cell membrane, proteolysis of cytoplasmic and nuclear proteins, and eventual destruction of nuclear DNA. Our laboratory is interested in the reversibility of apoptosis. We have initial evidence that DNA repair is activated early in p53-induced apoptosis and may be involved in its reversibility. The present work further strengthens our proposition that p53-inducedapoptosisisreversible. We show that p53 activation induces phosphatidylserine (PS) externalization early in apoptosis, and that these early apoptotic cells with externalized PS can be rescued and proliferate if the apoptotic stimulus is removed. In addition, we show that unscheduled DNA synthesis occurs in early apoptotic cells, and that if DNA repair is inhibited by aphidicolin, apoptosis is accelerated. These results confirm that early p53-induced apoptotic cells can be rescued from the apoptotic program, and that DNA repair can modulate that cell death process.Cell Death and Differentiation (2001) 8, 182 ± 191.

Cell and tissue growth is regulated through a complex interplay of stimulatory and inhibitory signals. We describe two biological actions of transforming growth factor beta 1 (TGF-beta 1) in primary cultures of rabbit uterine epithelial cells: (i) inhibition of cell proliferation and (ii) a concomitant increase in cells undergoing apoptosis (programmed cell death). It is proposed that proliferation and apoptosis together comprise normal cell growth regulation.

Abstractp53 is a complex molecule involved in apoptosis, cell cycle arrest, and DNA repair. Since apoptosis may play an important role in deletion of neoplastic cells, an understanding of the mechanism of p53-induced apoptosis may be critical for possible future therapeutic interventions. Recent evidence suggests that p53-induced apoptosis may involve members of the nucleotide excision repair (NER) family, linking these two cellular events. Our work using a temperature-sensitive p53 construct further analyzes p53-induced apoptosis in cultured murine mammary epithelial cells and also suggests that DNA repair plays a role in that process. Although p21 is induced in our system, apoptosis occurs without a detectable preceding G1 cell cycle arrest and independent of cellular alterations brought on by the temperature shift. In addition, clonogenic assays suggest that early stages of p53-induced apoptosis may be reversible upon removal of the apoptosis stimulus. As a possible explanation for this reversibility, our results show that general DNA repair activity increases early in p53-induced apoptosis. We also show that caspase-3 is activated at a timepoint when colony formation begins to drop, suggesting a possible mechanism for the point of no return in p53-induced apoptosis. Cell Death and Differentiation (2000) 7, 393 ± 401.

The study of growth of endometrial cells is of importance in reproductive biology. Several factors and hormones are thought to play important roles in the control of growth. Prostaglandin F2 alpha (PGF2 alpha) causes an increase in both tritiated thymidine ([3H]Tdr) incorporation into DNA and in the cell number of primary cultures of rabbit endometrial cells cultured in a serum-free, chemically defined media. Prostaglandins F1 alpha, E1, E2, A2, and B2 and arachidonic acid (all tested at 10(-7) M) do not affect [3H]Tdr incorporation as compared to control cultures. The increase in [3H]Tdr incorporation into DNA in response to PGF2 alpha stimulation is concentration-dependent (optimal approximately 3 X 10(-7) M) and is seen starting approximately 9 hr poststimulation. Both prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), but not PGs F1 alpha, I2, A2, B2, their parent molecules, or related molecules, antagonize and can completely block the PGF2 alpha-induced increase in [3H]Tdr incorporation into DNA. This antagonism is seen both when the cells are pretreated with PGE1 prior to the PGF2 alpha stimulation and when the cells are exposed to both PGE1 and PGF2 alpha simultaneously. Exogenously added 8-Br-cAMP mimics the PGE1 antagonism of PGF2 alpha. The PGF2 alpha-induced increase in [3H]Tdr incorporation is not synergistic, antagonistic, or additive with the [3H]Tdr incorporation increase in response to either estradiol-17 beta or epidermal growth factor. The specific effect of PGF2 alpha on primary culture endometrial cell growth and its antagonism by PGE1, PGE2, and 8-Br-cAMP are new findings.

Disease-associated BRCA2 mutations typically result in protein truncations that delete the phosphorylation-regulated S3291 BRCA2 domain that interacts with Rad51. BRCA2 hereditary breast cancers are usually ER+, differing from BRCA1 hereditary cancers, which are usually ER negative. We studied BRCA2 protein expression and S3291 phosphorylation in normal breast tissues and in sporadic breast cancers and observed that BRCA2 is expressed and phosphorylated in normal breast and 10 ER+ breast cancers but not in 10 ER negative breast cancers. In order to study this correlation between ER and BRCA2 expression we studied ER+ breast cancer cell lines. We found that a rapid increase in BRCA2 S3291 phosphorylation occurs following 17-beta-oestradiol (E2) treatment. This increase seen in BRCA2 total and phospho-S3291 protein levels was found to be unaffected with cycloheximide pre-treatment, but decreased following tamoxifen, ICI 182,780, or roscovitine treatment. This suggests a requirement for ER and cdk (cyclin-dependent kinase) in mediating the increased protein levels. MCF7 cell cycle distribution analysis following E2 both in the presence and absence of roscovitine (a cdk inhibitor) did not demonstrate any changes during an 8 hour period, which further supports our hypothesis that mitogenic effects of E2 are not predominant at early time points. Studies with MG132 proteasome inhibitor and siRNA to skp2 support a model in which skp2-mediated proteasomal degradation of BRCA2 rapidly degrades BRCA2 protein in the absence of hormone treatment which likely inhibits this pathway. E2 was shown to improve survival of MCF7 cells upon radiation treatment and roscovitine partially reversed this effect. We have demonstrated that BRCA2 protein is specifically expressed in ER+ breast cancers and are investigating a pathway that may show a link between E2 action and BRCA2 protein function in breast cancer.