PTEN deficiency: a role in mammary carcinogenesis.

The PTEN gene is often mutated in primary human tumors and cell lines, but the low rate of somatic PTEN mutation in human breast cancer has led to debate over the role of this tumor suppressor in this disease. The involvement of PTEN in human mammary oncogenesis has been implicated from studies showing that germline PTEN mutation in Cowden disease predisposes to breast cancer, the frequent loss of heterozygosity at the PTEN locus, and reduced PTEN protein levels in sporadic breast cancers. To assay the potential contribution of PTEN loss in breast tumor promotion, Li et al. [1] crossed Pten heterozygous mice with mouse mammary tumor virus-Wnt-1 transgenic (Wnt-1 TG, Pten+/-) mice. Mammary ductal carcinoma developed earlier in Wnt-1 TG, Pten+/- mice than in mice bearing either genetic change alone, and showed frequent loss of the remaining wild-type PTEN allele. These data indicate a role for PTEN in breast tumorigenesis in an in vivo model.

UVB induced cell cycle checkpoints in an early stage human melanoma line, WM35.

The activation of cell cycle checkpoints in response to genotoxic stressors is essential for the maintenance of genomic integrity. Although most prior studies of cell cycle effects of UV irradiation have used UVC, this UV range does not penetrate the earth's atmosphere. Thus, we have investigated the mechanisms of ultraviolet B (UVB) irradiation-induced cell cycle arrest in a biologically relevant target cell type, the early stage human melanoma cell line, WM35. Irradiation of WM35 cells with UVB resulted in arrests throughout the cell cycle: at the G1/S transition, in S phase and in G2. G1 arrest was accompanied by increased association of p21 with cyclin E/cdk2 and cyclin A/cdk2, increased binding of p27 to cyclin E/cdk2 and inhibition of these kinases. A loss of Cdc25A expression was associated with an increased inhibitory phosphotyrosine content of cyclin E- and cyclin A-associated cdk2 and may also contribute to G1 arrest following UVB irradiation. The association of Cdc25A with 14-3-3 was increased by UVB. Reduced cyclin D1 protein and increased binding of p21 and p27 to cyclin D1/cdk4 complexes were also observed. The loss of cyclin D1 could not be attributed to inhibition of either MAPK or PI3K/PKB pathways, since both were activated by UVB. Cdc25B levels fell and the remaining protein showed an increased association with 14-3-3 in response to UVB. Losses in cyclin B1 expression and an increased binding of p21 to cyclin B1/cdk1 complexes also contributed to inhibition of this kinase activity, and G2/M arrest. Oncogene (2000) 19, 4480 - 4490.

Stem cell factor inhibits erythroid differentiation by modulating the activity of G1-cyclin-dependent kinase complexes: a role for p27 in erythroid differentiation coupled G1 arrest.

Terminal erythroid differentiation is accompanied by decreased expression of c-Kit and decreased proliferation of erythroid progenitor cells. Using a newly established erythroleukemia cell line HB60-5, which proliferates in response to erythropoietin (Epo) and stem cell factor (SCF) and differentiates when stimulated with Epo alone, we characterized several events associated with the cell cycle during erythroid differentiation. Forty-eight h after SCF withdrawal and Epo stimulation, there was strong inhibition of cyclin-dependent kinase (cdk) 4 and cdk6 activities, associated with an increase in the binding of p27 and p15 to cdk6. A significant increase in the binding of p27 to cyclin E- and cyclin A-associated cdk2 correlated with the inhibition of these kinases. In addition, the expression of c-Myc and its downstream transcriptional target Cdc25A were found to be down-regulated during Epo-induced terminal differentiation of HB60-5 cells. The loss of Cdc25A was associated with an increase in the phosphotyrosylation of cyclin E-associated cdk2, which may contribute to cell cycle arrest during differentiation. Although overexpression of p27 in HB60-5 cells caused G1 arrest, it did not promote terminal erythroid differentiation. Thus, the cell cycle arrest that involves p27 is part of a broader molecular program during HB60-5 erythroid differentiation. Moreover, we suggest that SCF stimulation of erythroblasts, in addition to inhibiting erythroid differentiation, activates parallel or sequential signals responsible for maintaining cyclin/cdk activity.

Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.

Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Here we demonstrate that stable transfection and overexpression of the integrin-linked kinase (ILK), which interacts with the beta1 and beta3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. In addition, ILK overexpression results in the expression of p21 and p27 Cdk inhibitors with altered electrophoretic mobilities, with the p27 from ILK-overexpressing cells having reduced inhibitory activity. The transfer of serum-exposed IEC18 cells from adherent cultures to suspension cultures results in a rapid down-regulation of expression of cyclin D1 and cyclin A proteins as well as in retinoblastoma protein dephosphorylation. In marked contrast, transfer of ILK-overexpressing cells from adherent to suspension cultures results in continued high levels of expression of cyclin D1 and cyclin A proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These results indicate that, when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S cyclin-Cdk activities, which are normally regulated by cell adhesion and integrin engagement.

UVB radiation induces p21Cip1/WAF1 and mediates G1 and S phase checkpoints.

In a search for effectors and targets of UVB signaling in mammalian cells, we screened a keratinocyte cDNA library with differentially subtracted UVB-enriched cDNA probes. One of the UVB induced cDNA clones proved to be the rat p21Cip1/WAF1 homologue. UVB irradiation caused a rise in p53 protein levels, in association with induction of p21Cip1/WAF1 and cyclin G expression. The effects of UVB irradiation induced p21Cip1/WAF1 on the cell cycle were examined. In contrast to gamma irradiation, which caused G2 arrest, UVB treatment of asynchronous neonatal rat keratinocytes (NK) led to a marked inhibition of replicative DNA synthesis and prolonged G1 and S phase arrests, persisting to 18-24 h, with recovery of cycling by 36 h post-UVB. G1 arrest was accompanied by inhibition of cyclin D-, E- and A-associated kinases. Kinase inhibition was not due to reduction in cyclin or cdk proteins. While the association of cyclin E with Cdk2 was moderately reduced, cyclin D1/Cdk4 and cyclin A/Cdk2 complexes were not disrupted. The activating threonine 160 phosphorylation of Cdk2 in cyclin complexes was not inhibited. An incremental binding of p21 with Cdk4 paralleled the inhibition of cyclin D1/Cdk4 kinase and a similar rise in Cdk2 binding to p21 was associated with inhibition of cyclin E and cyclin A dependent kinases. Furthermore, a rise in measurable p21Cip1/WAF1-Cdk2 inhibitory activity paralleled the loss of G1 cyclin-dependent kinase activity, supporting a role for p21Cip1/WAF1 in the UVB-induced checkpoints.

Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone.

In the rat, transcripts encoding the myometrial gap junction protein, connexin-43 (Cx-43), increase dramatically with the onset of labor in association with an increase in the ratio of estrogen to progesterone in plasma. We examined whether the level of Cx-43 transcripts might be regulated by these steroids in the rat myometrium. Administration of progesterone to late pregnant rats abolished the more than 12-fold increase in transcripts seen in control rats at term and blocked delivery of the fetuses. Treatment of rats on day 15 of gestation (when Cx-43 mRNA levels are low) with the progesterone antagonist RU486 resulted in a significant (2.5-fold) increase in transcripts within 9 h, with the maximal (5.6-fold) increase occurring between 24-48 h, and preterm delivery occurring between 48-72 h. Administration of a single dose of 17 beta-estradiol (5 micrograms, sc) to nonpregnant rats resulted in a significant increase in Cx-43 transcripts within 3 h; these levels were increased 3-fold between 6-24 h, before falling to lower levels by 48 h. Progesterone (4 mg, sc) administration at the same time as estradiol significantly attenuated the estradiol response. Chronic estradiol administration (5 micrograms/12 h for 36 h) failed to maintain elevated levels of Cx-43 transcripts beyond 36 h. Administration of progesterone 12 h after estradiol prematurely reduced the level of Cx-43 transcripts. These data demonstrate that steroid hormones can modulate the level of steady state transcripts of Cx-43 during pregnancy in association with changes in uterine contractile activity. Furthermore, the data from the nonpregnant studies suggest that the levels of transcripts are regulated positively by estradiol and negatively by progesterone.

Increased expression of connexin-43 in the rat myometrium during labor is associated with an increase in the plasma estrogen:progesterone ratio.

The molecular mechanisms that regulate the synthesis of the myometrial gap junction protein, connexin-43 (Cx-43), are controversial. We measured myometrial Cx-43 messenger RNA, protein and gap junction frequency, and area in myometrial samples collected from nonpregnant rats and pregnant rats at days 5, 10, 15, 17, 18, 19, 20, 21, 22, 23 (during delivery), and 1 day postpartum and correlated these data with plasma concentrations of estradiol 17 beta and progesterone. Cx-43 transcripts were low or undetectable (connexin-43:glyceraldehyde phosphate dehydrogenase ratio < 0.2) in nonpregnant rats or in rats before day 10 of pregnancy. Transcripts rose to 0.52 +/- 0.11 on day 10, increased (2.9-fold) to 1.51 +/- 0.48 on day 22, and increased a further 2.9-fold to maximal levels of 4.42 +/- 0.67 during labor. Cx-43 protein was barely detectable on day 21 [0.12 +/- 0.04 relative optical density (ROD) units], increased 2.5-fold on day 22 (0.30 +/- 0.04 ROD units), and a further 3.7-fold during delivery (1.10 +/- 0.15 ROD units), at a time when gap junctions were present in large numbers in the cell membrane. Between day 21 and delivery the increase in Cx-43 transcripts (8.2-fold) and protein (9.2-fold) were of a similar magnitude. There was a significant positive correlation between the increases in Cx-43 transcripts and the increase in the ratio of plasma estradiol to progesterone. Levels of Cx-43 transcripts, protein, and gap junctions fell rapidly postpartum. Our data demonstrate: 1) that transcripts encoding the gap junction protein, Cx-43, are at maximal levels during delivery and that this increase is temporally associated with increases in Cx-43 protein and the appearance of gap junctions; and 2) that these data, in association with changes in plasma steroid concentrations, are consistent with myometrial Cx-43 transcript levels being regulated positively by estrogen and negatively by progesterone during pregnancy.