E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos.

The E2F transcription factors are thought to be key downstream targets of the retinoblastoma protein (pRB) tumor suppressor. It is widely believed that E2F1, E2F2, and E2F3 can all activate cellular proliferation but that E2F1 is the specific inducer of apoptosis. Here we show that the E2f3 mutation completely suppresses both the inappropriate proliferation and the p53-dependent apoptosis arising in the Rb mutant embryos. Through the analysis of Rb(-/-);E2f3(+/-) embryos, we have been able to separate E2F3's role in the induction of apoptosis from its ability to induce proliferation. Thus, contrary to the prevailing view of E2F action, E2F3 makes a major contribution to the apoptosis resulting from pRB loss.

Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors.

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death.

PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.

Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast.

Germline mutations in PTEN, encoding a dual-specificity phosphatase on 10q23.3, cause Cowden syndrome (CS), which is characterized by a high risk of breast and thyroid cancers. Loss of heterozygosity of 10q22-24 markers and somatic PTEN mutations have been found to a greater or lesser extent in a variety of sporadic component and noncomponent cancers of CS. Among several series of sporadic breast carcinomas, the frequency of loss of flanking markers around PTEN is approximately 30 to 40%, and the somatic intragenic PTEN mutation frequency is <5%. In this study, we analyzed PTEN expression in 33 sporadic primary breast carcinoma samples using immunohistochemistry and correlated this to structural studies at the molecular level. Normal mammary tissue had a distinctive pattern of expression: myoepithelial cells uniformly showed strong PTEN expression. The PTEN protein level in mammary epithelial cells was variable. Ductal hyperplasia with and without atypia exhibited higher PTEN protein levels than normal mammary epithelial cells. Among the 33 carcinoma samples, 5 (15%) were immunohistochemically PTEN-negative; 6 (18%) had reduced staining, and the rest were PTEN-positive. In the PTEN-positive tumors as well as in normal epithelium, the protein was localized in the cytoplasm and in the nucleus (or nuclear membrane). Among the immunostain negative group, all had hemizygous PTEN deletion but no structural alteration of the remaining allele. Thus, in these cases, an epigenetic phenomenon such as hypermethylation, -ecreased protein synthesis or increased protein degradation may be involved. In the cases with reduced staining, 5 of 6 had hemizygous PTEN deletion and 1 did not have any structural abnormality. Finally, clinicopathological features were analyzed against PTEN protein expression. Three of the 5 PTEN immunostain-negative carcinomas were also both estrogen and progesterone receptor-negative, whereas only 5 of 22 of the PTEN-positive group were double receptor-negative. The significance of this last observation requires further study.

Activation of posterior pair-rule stripe expression in response to maternal caudal and zygotic knirps activities.

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to the maternal concentration gradient of the anterior determinant BICOID and repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. Here we show that the zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. The results suggest that activation and the expression level mediated by the hairy stripe 6-element depend on the number of activator binding sites, likely to involve additive rather than synergistic interactions. We found an identical transacting factor requirement for hairy stripe 6 and 7 expression. The arrangement of the corresponding binding sites for the common factors involved in the control of the two stripes share a high degree of similarity, but some of the factors exert opposite regulatory functions within the two enhancer elements.

Linking Myb to the cell cycle: cyclin-dependent phosphorylation and regulation of A-Myb activity.

A-myb, a conserved member of the Myb proto-oncogene family, encodes a sequence-specific DNA binding protein (A-Myb) that binds to and transactivates promoters containing myb-binding sites. Previous work has suggested that the C-terminus of A-Myb functions as a regulatory domain, however, the physiological signals that control the activity of A-Myb have not yet been identified. The presence of potential phosphorylation sites for cyclin-dependent kinases in the C-terminus of A-Myb has prompted us to examine the possibility that the function of A-Myb is controlled by the cell cycle. We here show that the transactivation potential of A-Myb is repressed by the C-terminal domain and that phosphorylation of A-Myb, induced by cyclins A and E, relieves this inhibitory effect. Our work provides the first evidence that the function of A-Myb is regulated by the cell cycle machinery and that the carboxy-terminal domain of A-Myb acts as a cell cycle sensor. In addition, we show that A-myb mRNA expression is also cell cycle regulated and attains maximal levels during the late G1- and early S-phase. Thus, A-Myb appears to be controlled by two different mechanisms resulting in maximal A-Myb activity during the G1/S-transition and the S-phase of the cell cycle.

Phosphorylation and activation of B-Myb by cyclin A-Cdk2.

BACKGROUND: Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle. Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified. B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells. Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle. In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks. RESULTS: We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein. Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region. Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase. CONCLUSIONS: This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery. We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb. Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.