Canadian consensus recommendations on the management of venous thromboembolism in patients with cancer. Part 1: prophylaxis.

Patients with cancer are at increased risk of venous thromboembolism (vte). Anticoagulation therapy has been shown to prevent vte; however, unique clinical circumstances in patients with cancer can often complicate the decisions surrounding the administration of prophylactic anticoagulation. No national Canadian guidelines on the prevention of cancer-associated thrombosis have been published. We therefore aimed to develop a consensus-based, evidence-informed guideline on the topic. PubMed was searched for clinical trials and meta-analyses published between 2002 and 2013. Reference lists of key articles were hand-searched for additional publications. Content experts from across Canada were assembled to review the evidence and make recommendations. Low molecular weight heparin can be used prophylactically in cancer patients at high risk of developing vte. Direct oral anticoagulants are not recommended for vte prophylaxis at this time. Specific clinical scenarios, including renal insufficiency, thrombocytopenia, liver disease, and obesity can warrant modifications in the administration of prophylactic anticoagulant therapy. There is no evidence to support the monitoring of anti-factor Xa levels in clinically stable cancer patients receiving prophylactic anticoagulation; however, factor Xa levels could be checked at baseline and periodically in patients with renal insufficiency. The use of anticoagulation therapy to prolong survival in cancer patients without the presence of risk factors for vte is not recommended.

Canadian consensus recommendations on the management of venous thromboembolism in patients with cancer. Part 2: treatment.

Patients with cancer are at increased risk of venous thromboembolism (vte). Anticoagulation therapy is used to treat vte; however, patients with cancer have unique clinical circumstances that can often make decisions surrounding the administration of therapeutic anticoagulation complicated. No national Canadian guidelines on the management of established cancer-associated thrombosis have been published. We therefore aimed to develop a consensus-based, evidence-informed guideline on the topic. PubMed was searched for clinical trials and meta-analyses published between 2002 and 2013. Reference lists of key articles were hand-searched for additional publications. Content experts from across Canada were assembled to review the evidence and make recommendations. Low molecular weight heparin is the treatment of choice for cancer patients with established vte. Direct oral anticoagulants are not recommended for the treatment of vte at this time. Specific clinical scenarios, including the presence of an indwelling venous catheter, renal insufficiency, and thrombocytopenia, warrant modifications in the therapeutic administration of anticoagulation therapy. Patients with recurrent vte should receive extended (>3 months) anticoagulant therapy. Incidental vte should generally be treated in the same manner as symptomatic vte. There is no evidence to support the monitoring of anti-factor Xa levels in clinically stable cancer patients receiving prophylactic anticoagulation; however, levels of anti-factor Xa could be checked at baseline and periodically thereafter in patients with renal insufficiency. Follow-up and education about the signs and symptoms of vte are important components of ongoing patient care.

PLC gamma contributes to metastasis of in situ-occurring mammary and prostate tumors.

Phospholipase C-gamma (PLCgamma) has been implicated in tumor cell motility required for invasiveness and metastasis. Diminished tumor dissemination has been demonstrated in xenograft models, but studies in naturally-occurring tumors are lacking, having been limited by the timing of the interventions. Therefore, we generated mice that express a doxycycline (DOX)-inducible dominant-negative fragment of PLCgamma, PLCz; this approach avoids the in utero lethality caused by the absence of PLCgamma. As we targeted two de novo-occurring carcinomas of the mammary (MMTV-driven polyoma middle T antigen model, PyVmT) and prostate (TRAMP model) glands, we limited expression to these epithelial cells by driving DOX transactivator from the prostatein C3 promoter. This avoids the confounding variable of potentially abrogating motility in stromal and endothelial cells. These mice developed normally in the presence of DOX, except for limited mammary development if treated before 6 weeks and immaturity of the prostate gland if treated before 2 weeks of age. DOX-mediated induction of PLCz from age 8 to 16 weeks in PyVmT mice decreased the number of lung metastases by >10-fold (P<0.06) without a detectable effect on in situ tumor cell proliferation or tumor size. Lung metastases were also significantly decreased in the TRAMP model in which the mice expressed the PLCz fragment (P<0.05). DOX treatment itself had no effect on tumor size or metastasis in control mice, nor did it affect tumor dissemination in nontransgenic littermates. In conclusion, abrogation of the PLCgamma signaling pathway can limit the metastatic potential of carcinomas.

castor encodes a novel zinc finger protein required for the development of a subset of CNS neurons in Drosophila.

Using an enhancer detection screen, we have identified castor, a new gene required for embryonic CNS development in Drosophila. Embryos that lack castor expression have a diminished CNS axonal network and express engrailed aberrantly late in CNS development. castor is unique among the previously described genes involved in Drosophila neurogenesis in that its expression is restricted to a subset of delaminated CNS neuroblasts and to ventral midline glial precursor cells. The putative castor gene product contains a novel zinc-binding domain and multiple transcriptional activation domains, suggesting that it acts as a transcription factor necessary for the development of a subset of CNS neuronal precursors.

Non-traumatic necrosis of bone (osteonecrosis) is associated with endothelial cell activation but not thrombophilia.

OBJECTIVE: The pathophysiology of non-traumatic osteonecrosis (ON) or avascular necrosis (AVN) of the femoral head remains poorly understood. Some studies have suggested the contribution of underlying thrombophilia as a mechanism; however, no specific thrombophilic factor has been consistently found in association with the disease. We are presenting data suggesting a role for endothelial cell activation rather than thrombophilia in ON. METHODS: A prospective consecutive cohort of 49 patients with a diagnosis of ON. The disease was considered idiopathic in five and secondary in 44 patients. The investigation included a coagulation and thrombophilia profile, endothelial cell activation and non-specific inflammatory markers as well as a biochemical profile. Statistical analysis using Fisher's exact test was obtained to assess correlation between endothelial cell markers and variables of inflammation. RESULTS: Patients with non-traumatic ON were not found to have a specific underlying thrombophilic factor compared with the general population. Out of 49 patients,19 had elevation of at least one endothelial cell markers. We found that activation of endothelial cell markers was independently correlated to ON but not correlated to the presence of inflammation (P = 1.0000). CONCLUSION: These results suggest that non-traumatic ON is not associated with a specific thrombophilic abnormality in those affected. This study demonstrates a potential association between regional endothelial dysfunction and ON. More studies are needed at a molecular level to further investigate the specific role of endothelium in the physiopathology of ON. A better understanding of the underlying mechanism could lead to potential preventive and therapeutic strategies of this devastating disease.

Evolutionary conservation of homeodomain-binding sites and other sequences upstream and within the major transcription unit of the Drosophila segmentation gene engrailed.

The engrailed (en) gene functions throughout Drosophila development and is expressed in a succession of intricate spatial patterns as development proceeds. Normal en function relies on an extremely large cis-acting regulatory region (70 kilobases). We are using evolutionary conservation to help identify en sequences important in regulating patterned expression. Sequence comparison of 2.6 kilobases upstream of the en coding region of D. melanogaster and D. virilis (estimated divergence time, 60 million years) showed that 30% of this DNA occurs in islands of near perfect sequence conservation. One of these conserved islands contains binding sites for homeodomain-containing proteins. It has been shown genetically that homeodomain-containing proteins regulate en expression. Our data suggested that this regulation may be direct. The remaining conserved islands may contain binding sites for other regulatory proteins.

Electron microscopic heteroduplex mapping identifies regions of the engrailed locus that are conserved between Drosophila melanogaster and Drosophila virilis.

Physical localization of mutations in the engrailed (en) gene suggested that at least 70 kilobases (kb) of genomic sequences contribute to the normal function of this gene. Molecular characterization has suggested that en function is encoded in a small, 4.5-kb primary transcript. To identify functional regions within the 70 kb of the en locus of D. melanogaster, we identified sequences conserved in the D. virilis genome (estimated divergence time, 60 million years). Based on homology to D. melanogaster, we isolated en DNA from a D. virilis genomic library. Electron microscopic heteroduplex analysis indicated that in 70 kb there is 20 kb of conserved DNA in 33 different regions dispersed throughout the en locus, including two which encode parts of the major embryonic transcript. The conserved regions are in the same linear order and are spaced by similar lengths of nonconserved sequences in the D. virilis and D. melanogaster DNAs. What functional constraints have enforced conservation of sequences throughout the entire 70 kb and protected the region from divergence of size and arrangement? Our working hypothesis is that sequences necessary for the complex spatial and temporal pattern of en expression are dispersed throughout the 70-kb en locus and that selection for proper regulation restricts evolutionary divergence.

Dolichol-phosphate-mannose-3 (DPM3)/prostin-1 is a novel phospholipase C-gamma regulated gene negatively associated with prostate tumor invasion.

The most ominous development in tumor progression is the transition to an invasive and metastatic phenotype. Little is known, however, about the molecular alterations that cause a tumor to become invasive. In view of this, we have used microarray expression analysis to evaluate the expression profiles of a unique panel of human DU145 prostate cancer sublines that vary in their invasive potential. The three DU145 sublines expressed epidermal growth factor (EGF) receptors that differed in their ability to activate phospholipase C-gamma (PLC gamma). Three-way analyses yielded 11 genes out of 4608 genes screened that associated directly or inversely with invasive potential. The gene whose expression correlated most strongly with lack of invasion was identified as a potential invasion suppressor and called prostin-1. Pharmacological inhibition of PLC gamma (U73122) confirmed that PLC gamma signaling suppressed prostin-1 in that U73122 treatment caused induction of prostin-1 in PLC gamma competent cells. The prostin-1 gene, conserved through phylogeny, is induced by androgen in LNCaP cells and encodes a 92 amino acid protein. The protein shares no extensive homologies with other known genes, yet was recently identified as a small stabilizer subunit of the dolichol-phosphate-mannose (DPM) synthase complex. That DPM3/prostin-1 might suppress tumor progression was supported by the finding that exogenous expression in COS cells leads to apoptosis. These findings support the use of model cell lines to identify putative tumor suppressors and promoters.

Predictors of the post-thrombotic syndrome during long-term treatment of proximal deep vein thrombosis.

BACKGROUND: The post-thrombotic syndrome is a chronic, poorly understood complication of deep venous thrombosis (DVT). OBJECTIVES: To evaluate predictors of the post-thrombotic syndrome, including intensity of long-term anticoagulation, and to assess the impact of the post-thrombotic syndrome on quality of life. PATIENTS AND METHODS: The setting was 13 Canadian hospitals and one US hospital. One hundred and forty-five patients with an unprovoked episode of proximal DVT who were initially treated with 3 months of conventional-intensity warfarin [target International Normalized Ratio (INR) of 2.5] then participated in a trial comparing two intensities of long-term warfarin therapy (target INR 2.5 vs. INR 1.7). Post-thrombotic syndrome was assessed at the end of the trial using a validated clinical scale. Generic and venous disease-specific quality of life was compared in patients with and without the post-thrombotic syndrome. Multivariable regression analyses were performed to identify predictors of the post-thrombotic syndrome and of its severity. RESULTS: After an average follow-up of 2.2 years, the prevalence of post-thrombotic syndrome was 37% and of severe post-thrombotic syndrome was 4%. Quality of life was worse in patients with the post-thrombotic syndrome compared with patients who did not have it. The presence of factor (F)V Leiden or the prothrombin gene mutation was an independent predictor of both a lower risk (P = 0.006) and reduced severity (P = 0.045) of the post-thrombotic syndrome. Intensity of anticoagulation did not influence the risk of developing the post-thrombotic syndrome. CONCLUSIONS: The post-thrombotic syndrome is a frequent and burdensome complication of proximal DVT, even among patients maintained on long-term oral anticoagulation. While the presence of FV Leiden or prothrombin gene mutation appears to be associated with a reduced risk of post-thrombotic syndrome, this finding requires further evaluation in prospective studies.

Unusual properties of regulatory DNA from the Drosophila engrailed gene: three "pairing-sensitive" sites within a 1.6-kb region.

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called "pairing-sensitive sites" (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter.

A fragment of engrailed regulatory DNA can mediate transvection of the white gene in Drosophila.

We have found a fragment of engrailed regulatory DNA that has an unusual effect on expression of a linked marker gene, white, in the P element transposon CaSpeR. Normally, flies homozygous for a given CaSpeR insertion have darker eyes than heterozygotes. However, when a particular engrailed DNA fragment is included in that transposon, homozygotes often have lighter eyes than heterozygotes. Thus, engrailed DNA appears to cause white expression to be repressed in homozygotes. The suppression of white is dependent on the proximity of the two transposons in the genome-either in cis (i.e., on the same chromosome) or in trans (i.e., on homologous chromosomes). Thus, the engrailed fragment is mediating a phenomenon similar to that mediated by the zeste gene at the white locus. However, the interactions we observe do not require, nor are influenced by, mutations of zeste. We suggest that the engrailed DNA contains one or more binding sites for a protein that facilitates interactions between transposons. The normal function of these sites may be to mediate interactions between distant cis-regulatory regions of engrailed, a large locus that extends over 70 kilobases.

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes.

Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene (Boulay et al., 1987). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) (Sargent and Bennett, 1990). We have named the Drosophila snail-related gene escargot (esg), and the region of sequence conservation common to all three genes the 'snailbox'. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues, escargot is allelic with l(2)35Ce, an essential gene located near snail in the genome.

Molecular inhibition of phospholipase cgamma signaling abrogates DU-145 prostate tumor cell invasion.

Up-regulated signaling from the epidermal growth factor receptor (EGFR) has been correlated with tumor invasion and metastasis in numerous human neoplasias. Recently, we have demonstrated that increased levels of EGFR promote the invasiveness of human prostate carcinoma DU-145 cells. However, the intracellular signaling pathway responsible for this enhanced tumor invasiveness has not been identified. We postulated that increased cell motility signaled via phospholipase Cgamma (PLCgamma) activation was critical for tumor invasiveness. Highly invasive DU-145 cells engineered to overexpress the EGFR were stably transfected with a dominant-negative fragment of PLCgamma from the Z-region (PLCz) or with irrelevant peptide minigenes. PLCz was expressed only in the appropriate transfectant lines, with a concomitant decrease in inositol phosphate generation. The transfectant cell lines all formed tumors when inoculated into the peritoneal cavity of athymic mice. Tumors from the cells expressing PLCz fragment were significantly less invasive than the transfectants containing the control minigenes, as assessed by the diaphragm invasion model and invasion into abdominal soft organs. The cells expressing PLCz grew and formed colonies in soft agar at rates comparable to the cells expressing the control minigenes. These data suggest that up-regulated signaling by EGFR promotes prostate tumor invasiveness secondary to increased cell motility. Furthermore, PLCgamma represents a potential therapeutic target to limit tumor progression promoted by up-regulated signaling from the EGFR and related receptors with intrinsic tyrosine kinase activity.

A role for phospholipase C-gamma-mediated signaling in tumor cell invasion.

The invasive and metastatic transformation of cancers often results in death. However, the mechanisms that promote this transformation remain unclear. Two closely related receptors, the epidermal growth factor receptor (EGFR) and ErbB2, are overexpressed in a significant percentage of breast and prostate carcinomas, among others, with this up-regulated signaling correlating with tumor progression. Previous studies in our laboratory have demonstrated that an EGFR-phospholipase C (PLC)gamma-mediated motility-associated signaling pathway is rate-limiting for tumor cell invasion in vitro and in vivo in one model of prostate carcinoma. Therefore, we investigated whether this PLCgamma signaling pathway also was rate-limiting for invasion in other tumor cell lines and types and whether this EGFR activity is subsumed by the closely related ErbB2. We determined the effects of PLCgamma signal abrogation by pharmacological (U73122) and molecular (expression of the dominant-negative PLCz) means on the in vitro invasiveness of tumor cells. Inhibition of PLCgamma signaling concomitantly decreased invasiveness of de novo-occurring transgenic adenocarcinoma mouse prostate (TRAMP) lines and the human breast cancer cell lines MDA-468 and MDA-231; these lines present up-regulated EGFR signaling. Because the prostate and breast cancer lines usually present autocrine stimulatory loops involving EGFR, we also examined transgenic adenocarcinoma mouse prostate C1 and MDA-468 treated with the EGFR-specific kinase inhibitor PD153035 to determine whether invasiveness is dependent on EGFR signaling. PD153035 reduced invasiveness to levels similar to those seen with U73122, suggesting that the autocrine EGFR stimulatory loop is functioning to promote invasiveness. To determine whether this signaling pathway also promotes invasiveness of ErbB2-overexpressing tumors, we examined the human breast carcinoma line MDA-361; again, U73122 inhibition of PLCgamma decreased invasiveness. In all situations, the inhibition of PLCgamma signaling did not decrease mitogenic signaling. Thus, the motility-associated PLCgamma signaling pathway is a generalizable rate-limiting step for tumor cell progression.

Malignant effusions are sources of fibronectin and other promigratory and proinvasive components.

Metastatic dissemination is the primary cause of death in ovarian cancer (OvCa) patients, and dissemination to pleural and peritoneal effusions is a common clinical event. Effusion samples were collected from 15 OvCa patients. Twenty-six samples were collected prospectively, two were archival, and eight were taken from patients with other malignancies. Twenty-nine samples were from malignant ascites, and seven specimens were pleural fluids. In addition, six ascites and two pleural fluids from noncancer patients were studied as effusion controls. Effusion supernatants were tested for migration-stimulation activity, using A2058 human melanoma cells as the index responder cell. Malignant samples induced a 400-1200% increase in migration. Sixty percent of the migration was inhibited by incubation of the malignant fluid with antifibronectin (FN) antibody, in contrast to 75% inhibition of control fluid-stimulated migration (P = 0.017). Gelatin zymography and Western blot analyses showed that latent and activated MMP-2 and MMP-9 collagenases, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were present in all malignant fluids. Serial samples were taken from several patients, and a trend for correlation between MMPs and clinical behavior of the tumors was shown. Free TIMP-2 correlated with CA-125 levels in two patients for whom serial samples were available. The demonstration of promigratory and proinvasive activity in malignant effusions is consistent with their association with other metastatic disease in OvCa patients and their function as a haven for metastatic cells.

Inflammation markers and their trajectories after deep vein thrombosis in relation to risk of post-thrombotic syndrome.

BACKGROUND: Post-thrombotic syndrome (PTS) is a frequent chronic complication of deep vein thrombosis (DVT). OBJECTIVE: In the BioSOX study, we investigated whether inflammation markers predict the risk of PTS after DVT. METHODS: We measured C-reactive protein (CRP), ICAM-1, interleukin (IL)-6, and IL-10, at baseline, and 1 month and 6 months after a first proximal DVT, among 803 participants in the SOX trial. Participants were prospectively followed for 24 months for development of PTS. RESULTS: Median CRP levels at 1 month, ICAM-1 levels at baseline, 1 month and 6 months, IL-6 levels at 1 month and 6 months and IL-10 levels at 6 months were higher in patients who developed PTS than in those who did not. Multivariable regression with the median as a cutoff showed risk ratios (RRs) for PTS of 1.23 (95% confidence interval [CI] 1.05-1.45) and 1.25 (95% CI 1.05-1.48) for ICAM-1 at 1 month and 6 months, respectively, and 1.27 (95% CI 1.07-1.51) for IL-10 at 6 months. Quartile-based analysis demonstrated a dose-response association between ICAM-1 and PTS. ICAM-1 and IL-10 were also associated with PTS severity. Analysis of biomarker trajectories after DVT demonstrated an association between the highest-trajectory group of ICAM-1 and PTS. CONCLUSIONS: In this prospective study, ICAM-1 over time was most consistently associated with the risk of PTS. Further study is required to confirm these findings and assess their potential clinical relevance.

Estrogen receptors in cultured rat uterine cells: induction of progesterone receptors in the absence of estrogen receptor processing.

When cultured rat uterine cells were treated for up to 6 h with 5 nM 17 beta-estradiol, no decrease in the [3H] estradiol-binding capacity of the cells was observed (i.e. no processing). This was true whether the cells were treated directly with 5 nM [3H]estradiol or with 5 nM unlabeled 17 beta-estradiol followed by homogenization and exchange with [3H]estradiol in vitro. In additional experiments, intact cells were treated with medium containing 5 nM [3H]estradiol for 30 min, and then that medium was removed and replaced with medium containing 5 nM unlabeled 17 beta-estradiol. Receptor-bound estradiol in intact cells was totally exchangeable with estradiol in the culture medium (t1/2, approximately 90 min). Six-hour treatment of cells with 5 nM 17 beta-estradiol led to a 50% increase in the [3H]progesterone-binding capacity of the cells, while no loss of estrogen-binding capacity occurred. These results indicate that progesterone receptors can be induced by estrogen in the rat uterus in the absence of estrogen receptor processing.

Estrogen receptors in rat uterine cell cultures: effects of medium on receptor concentration.

Both the estrogen responsiveness and -binding capacity of cultured rat uterine cells were decreased dramatically when the medium was not changed at 24-h intervals. Treatment of cells for 24 h with 1 nM 17 beta-estradiol in fresh medium led to a 3-fold increase in progesterone receptor concentration, but without fresh medium, no increase in progesterone receptors was observed. When the medium was changed on cells with a low estrogen-binding capacity (depleted cells), a 6- to 10-fold increase in estrogen-binding capacity (to in vivo levels) occurred within 24 h (fed cells), and total protein was increased 2-fold. The high and low affinity binding characteristics of fed and depleted cells were identical. Recovery of the estrogen-binding capacity of depleted cells was relatively slow, increasing after a 6-h lag and reaching maximal levels by 24 h. While 6 h of 10(-5) M cycloheximide treatment (protein synthesis inhibited greater than 95%) had little effect on control estrogen binding levels, it completely inhibited the increase in the estrogen-binding capacity induced by changing the medium on depleted cells. These results indicate that estrogen-binding activity can be varied in cultured rat uterine cells by changing medium conditions and suggest that these changes are due to differences in receptor protein levels and not to a receptor activation-inactivation phenomenon.

Primary cultures of estrogen-responsive cells from rat uteri: induction of progesterone receptors and a secreted protein.

Uterine cells from immature rats can be grown in culture, are estrogen responsive, and contain estrogen receptors. Progesterone receptor is induced within 1 day of 17 beta-estradiol treatment, with maximal response occurring after 2 days of treatment (300-500% of the control value). Induction of progesterone receptor occurred at physiological 17 beta-estradiol concentrations, with half-maximal response at about 5 X 10(-11) M. 17 beta-Estradiol induced the synthesis of a secreted protein (mol wt, 130,000) in a dose-dependent fashion. This 130-K protein was also induced by 16 alpha-estradiol (1-10 nM), but not by progesterone (10 nM), testosterone (1 nM), or dexamethasone (1 nM). Examination of the estrogen-binding properties of the cultured cells shows a saturable binding site (Kd approximately 10(-10) M) which can be translocated to the nucleus. Estrogen receptors were maintained at in vivo levels as uterine cells proliferated throughout 10 days of culture. This was in contrast to estrogen receptor levels in Fischer 344 rat pituitary cell cultures, which dropped off drastically on a DNA basis as cells proliferated. These studies indicate that estrogen receptor-containing rat uterine cells proliferate and are responsive in primary cell cultures.