Pluto's tenuous nitrogen atmosphere was first detected by the imprint left on the light curve of a star that was occulted by the planet in 1985 (ref. 1), and studied more extensively during a second occultation event in 1988 (refs 2-6). These events are, however, quite rare and Pluto's atmosphere remains poorly understood, as in particular the planet has not yet been visited by a spacecraft. Here we report data from the first occultations by Pluto since 1988. We find that, during the intervening 14 years, there seems to have been a doubling of the atmospheric pressure, a probable seasonal effect on Pluto.

Progesterone in hormonal preparations increases the incidence of breast cancer. Tissue factor (TF), the initiator of the extrinsic coagulation pathway, is associated with metastasis in a wide variety of cancers. We demonstrate herein that TF mRNA and protein are up-regulated by progesterone in the breast cancer cell line ZR-75. Epidermal growth factor, also associated with increased breast cancer risk, did not regulate TF. The increase in TF is both rapid and transient; increasing after 6 h, reaching a maximum at 24 h, before decreasing to basal levels at 72 h. Sucrose gradient experiments demonstrated that TF is located in the heavy fraction of the plasma membrane, although caveolin-1 is not expressed in ZR-75. To understand the physiological implications of an increase in TF, we performed coagulation and invasion assays. An increase in TF corresponded to an increase in procoagulant activity. Furthermore, progesterone increased the invasion of ZR-75 cells through a matrigel, an effect that was blocked by an antibody against TF. Because TF expression is associated with an enhanced risk of metastasis, we postulate that the progesterone-dependent up-regulation of TF provides a survival advantage to burgeoning breast cancer cells and may contribute to the increased risk of cancer associated with combined hormone replacement therapy.

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.

Estrogen replacement therapy and other unopposed estrogen treatments increase the incidence of endometrial abnormalities, including cancer. However, this effect is counteracted by the co-administration of progesterone. In the endometrium, glucose transporter (GLUT) expression and glucose transport are known to fluctuate throughout the menstrual cycle. Here, we determined the effect of estrogen and progesterone on the expression of GLUT1-4 and on the transport of deoxyglucose in Ishikawa endometrial cancer cells. Cells were incubated with estrogen, progesterone or combined estrogen and progesterone for 24 h and the effect on the expression of GLUT1-4 and on deoxyglucose transport was determined. We show that GLUT1 expression is upregulated by estrogen and progesterone individually, but that combined estrogen and progesterone treatment reverses this increase. Hormonal treatments do not affect GLUT2, GLUT3 or GLUT4 expression. Transport studies demonstrate that estrogen increases deoxyglucose transport at Michaelis-Menten constants (K m s) corresponding to GLUT1/4, an effect which disappears when progesterone is added concomitantly. These data demonstrate that different hormonal treatments differentially regulate GLUT expression and glucose transport in this endometrial cancer cell line. This regulation mirrors the role played by estrogen and progesterone on the incidence of cancer in this tissue and suggests that GLUT1 may be utilized by endometrial cancer cells to fuel their demand for increased energy requirement.

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.