Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition.

Recent findings have established a connection between DNA methylation and transcriptionally inactive chromatin characterized by deacetylated histones. Because the absence of estrogen receptor alpha (ERalpha) gene expression has been associated with aberrant methylation of its CpG island in a significant fraction of breast cancers, we tested whether histone deacetylase activity contributes to the transcriptional inactivation of the methylated ER gene in a panel of ER-negative human breast cancer cells. Treatment of these cells with trichostatin A, a specific histone deacetylase inhibitor, led to dose- and time-dependent re-expression of ER mRNA as detected by reverse transcription-PCR without alteration in ERalpha CpG island methylation. Trichostatin A-induced ER re-expression was associated with increased sensitivity to DNase I at the ER locus in MDA-MB-231 cells. These data implicate inactive chromatin mediated by histone deacetylation as a critical component of ER gene silencing in human breast cancer cells. Therefore, histone deacetylation may be a potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers.

Mapping of ER gene CpG island methylation-specific polymerase chain reaction.

Southern analysis has shown that DNA from 25% of primary estrogen receptor (ER) alpha-negative breast tumors displays aberrant methylation at one site within the ER gene CpG island. To examine more sites and increase sensitivity, we developed a methylation-specific PCR assay to map methylation of the entire ER CpG island. The island was unmethylated in normal breast tissue and ER-positive breast cancer cell lines, but extensively methylated in all ER-negative cell lines and breast tumors examined. In addition, some of the ER-positive/progesterone receptor-negative and ER-positive/progesterone receptor-positive tumors (about 70% and 35%, respectively) displayed methylation of the ER CpG island, suggesting that this heterogeneity within tumor cell populations could potentially shed light on the etiology of ER-negative recurrent tumors arising from ER-positive tumors.

Aberrant methylation of the estrogen receptor and E-cadherin 5' CpG islands increases with malignant progression in human breast cancer.

Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.

Cooperation of TGF alpha and c-Myc in mouse mammary tumorigenesis: coordinated stimulation of growth and suppression of apoptosis.

We have previously shown that TGF alpha and c-Myc interact in a strong, synergistic fashion to induce mammary gland tumors in double transgenic mice. Here we show this interaction can be explained, at least in part, by a cooperative growth stimulus by the two proteins, and by TGF alpha-mediated inhibition of c-Myc-induced apoptosis. We initially compared rapidly progressing mammary tumors from double transgenic mice to long latency tumors from single transgenic mice and observed a striking difference in the occurrence of apoptosis among the three groups. Tumors exhibiting apoptosis were derived exclusively from mice that expressed the c-myc transgene in the absence of the TGF alpha transgene, indicating that TGF alpha might protect c-Myc-overexpressing cells from programmed cell death. Cell lines were derived from single and double transgenic mammary tumors to examine further the mechanism underlying the cooperative interaction between the two gene products. In accordance with our in vivo data, apoptosis was only detected when the c-myc transgene was expressed without the TGF alpha transgene. Furthermore, exogenous addition of TGF alpha inhibited apoptosis in cells overexpressing c-Myc alone. In addition, tumor-derived cells that overexpressed both TGF alpha and c-Myc exhibited faster growth rates in vitro and in vivo and were less sensitive to the inhibitory effects of TGF beta in vitro compared to cell lines expressing only one of the transgenes. Based on our findings we propose that TGF alpha acts both as a proliferative and a survival factor for c-Myc-expressing tumor cells. Our results indicate that TGF alpha and c-Myc cooperate in tumorigenesis via a dual mechanism: TGF alpha can inhibit c-Myc-induced apoptosis and both proteins provide a growth stimulus.

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells.

Estrogen receptor (ER)-negative breast cancer cells display extensive methylation of the ER gene CpG island and elevated DNA methyltransferase (DMT) expression compared to ER-positive cells. The present study demonstrates that DMT protein levels tightly correlate with S phase fraction in ER-positive cells, whereas ER-negative cells express DMT throughout the cell cycle. In addition, levels of p21CIP1, which disrupts DMT binding to PCNA, are inversely correlated with DMT levels. Therefore increased DMT expression in ER-negative cells is not simply due to elevated S-phase fraction, but rather to more complex changes that allow cells to escape normal cell cycle-dependent controls on DMT expression. Because ER-negative breast tumors often have activated growth factor pathways, the impact of these pathways on DMT expression was examined in ER-positive cells. Stable transfection with fibroblast growth factors (FGFs) 1 and 4 led to increased DMT expression that could not be accounted for by a shift in S phase fraction. Elevated DMT protein expression in FGF-transfectants was accompanied by a significant decrease in p21, again suggesting a reciprocal relationship between these two proteins. However, acquisition of an estrogen-independent phenotype, even in conjunction with elevated DMT levels, was not sufficient to promote ER gene silencing via methylation. These results indicate that multiple steps are required for de novo methylation of the ER CpG island.

Epidermal growth factor-dependent cell cycle progression is altered in mammary epithelial cells that overexpress c-myc.

Amplification and overexpression of the c-myc gene are common in primary human breast cancers and have been correlated with highly proliferative tumors. Components of the epidermal growth factor (EGF) receptor signaling pathway are also often overexpressed and/or activated in human breast tumors, and transgenic mouse models have demonstrated that c-myc and transforming growth factor alpha (a member of the EGF family) strongly synergize to induce mammary tumors. These bitransgenic mammary tumors exhibit a higher proliferation rate than do tumors arising in single transgenics. We, therefore, chose to investigate EGF-dependent cell cycle progression in mouse and human mammary epithelial cells with constitutive c-myc expression. In both species, c-myc overexpression decreased the doubling time of mammary epithelial cells by approximately 6 h, compared to parental lines. The faster growth rate was not due to increased sensitivity to EGF but rather to a shortening of the G1 phase of the cell cycle following EGF-induced proliferation. In cells with exogenous c-myc expression, retinoblastoma (Rb) was constitutively hyperphosphorylated, regardless of whether the cells were growth-arrested by EGF withdrawal or were traversing the cell cycle following EGF stimulation. In contrast, the parental cells exhibited a typical Rb phosphorylation shift during G1 progression in response to EGF. The abnormal phosphorylation status of Rb in c-myc-overexpressing cells was associated with premature activation of cdk2 kinase activity, reduced p27 expression, and early onset of cyclin E expression. These results provide one explanation for the strong tumorigenic synergism between deregulated c-myc expression and EGF receptor signal transduction in the mammary tissue of transgenic mice. In addition, they suggest a possible tumorigenic mechanism for c-myc deregulation in human breast cancer.

Detection of cyclin messenger RNAs by nonradioactive ribonuclease protection assay: a comparison of four detection methods.

Stable, nonradioactive riboprobes were used in ribonuclease protection assays to monitor the changes in cyclin mRNA expression during cell cycle progression in human mammary epithelial cells. Probes labeled with biotin demonstrated sufficient sensitivity (comparable to 32P) to detect these low-abundance mRNAs and thus offer a safe and easy alternative to traditional radioactive assays. Three detection systems based on chemiluminescence generated by horseradish peroxidase or alkaline phosphatase were compared for sensitivity, background and ease of use.

The biology of breast cancer.

This article focuses on the major hormones and growth factors for which a critical role in normal mammary growth has been clearly defined. Certainly other hormonal systems and growth factors could also affect breast cancer initiation and progression, but their exact contribution to normal and/or malignant breast cell growth is poorly delineated. Examples of such factors include somatostatin, mammostatin, mammary-derived growth inhibitor (MDGI), mammary-derived growth factor-1 (MDGF-1), inhibins, activins, androgens, glucocorticoids, vitamin D, thyroid hormones, ecosinoids, and oxytocin. Clearly, the hormonal regulation of breast cancer cell growth and survival is multifaceted and very complex. In particular, the effects of estrogens and anti-estrogens on breast cells may depend on their interaction with a wide variety of other pathways. In addition, these interactions may vary among individual breast tumors depending on other genetic changes in the tumor cells that have not been discussed here, such as oncogene activation and loss of tumor suppressors. A more detailed understanding of how cells circumvent a dependency on these pathways is greatly needed in order to identify new biological targets and to design novel therapies for breast cancers that are resistant to anti-estrogen therapy. Such agents could be used alone or in combination with anti-estrogens to improve response to a second course of hormonal therapy.

A survey of athletic medicine outreach programs in wisconsin.

In 1985, Sherman (21) reported a lack of organized and supervised health care for athletes in southern Wisconsin high schools and proposed that outreach athletic trainer services could help fill this gap. In order to determine the extent to which outreach programs are providing athletic training services, in January 1990, I surveyed 30 athletic medicine outreach services in Wisconsin to observe the frequency and scope of care being provided. Twenty-six surveys were returned. Results indicated that: (1) nearly half the high schools in Wisconsin subscribe to an outreach service; (2) outreach visits vary per individual school/service agreements, but average 2.5 visits per week, 2 hours per visit; (3) generally, outreach services furnish medical coverage to community athletic events as well as provide a source of continuing education programs for local coaches and administrators; (4) fees for services vary per contract, with nearly one-third of the respondents operating on a "no fee" volunteer basis; (5) nearly two-thirds of the outreach services emanate from a physical therapy department; and (6) 88% of services employ certified athletic trainers to provide outreach care. Outreach services appear as the mode of choice for providing athletic medicine in Wisconsin. Although some care is better than none, the quality and comprehensiveness of this care requires further investigation.

Defining a role for c-Myc in breast tumorigenesis.

The proto-oncogene c-myc is commonly amplified and overexpressed in human breast tumors, and the tumorigenic potential of c-myc overexpression in mammary tissue has been confirmed by both in vitro and in vivo models of breast cancer. However, the mechanisms by which Myc promotes tumorigenesis are not well understood. Recent evidence indicates that Myc can promote cell proliferation as well as cell death via apoptosis. These studies provide new insight and impetus in defining a role for c-Myc in breast tumorigenesis and may point toward novel targets for breast cancer therapy.

The genetic map around the tail kinks (tk) locus on mouse chromosome 9.

Tail kinks (tk) is a classical mouse skeletal mutation, located on Chromosome (Chr) 9. As the first step for the positional cloning of the tk gene, we have established a genetic map of a region surrounding the tk locus by generating a backcross segregating for tk. From this backcross, 1004 progeny were analyzed for the coat-color phenotype of the proximally located dilute (d) gene and for the distally flanking microsatellite marker, D9Mit12. Fifty-six recombinants between d and tk and 75 recombinants between tk and D9Mit12 were identified, completing a panel of 130 recombinants including one double recombinant. This panel allowed us to map five microsatellite loci as well as d and Mod-1 with respect to tk. We show that one of the microsatellite markers mapped, D9Mit9, does not recombine at all with tk in our backcross. This indicates that the D9Mit9 locus will serve as a good starting point for a chromosomal walk to the tk gene.

The loss of estrogen and progesterone receptor gene expression in human breast cancer.

Hormone responsiveness is a critical determinant of breast cancer progression and management, and the response to endocrine therapy is highly correlated with the estrogen receptor (ER)3 and progesterone receptor (PR) status of tumor cells. Thus, key areas of study in breast cancer are those mechanisms that regulate ER and PR expression in normal and malignant breast tissues. One-third of all breast cancers lack ER and PR; these conditions are associated with less differentiated tumors and poorer clinical outcome. In addition, approximately one-half of ER-positive tumors lack PR protein and patients with this phenotype are less likely to respond to hormonal therapies than those whose tumors express both receptors. Since PR is induced by ER; its presence is a marker of a functional ER. In this review, we will discuss possible mechanisms for loss of ER and PR gene expression, especially structural changes within each gene including deletions, polymorphisms or methylation. Improved understanding of the pathways that lead to loss of ER and/or PR proteins should allow the development of better predictive indicators as well as novel therapeutic approaches to target these hormone-independent cancers.

Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent.

Heparin binds to bovine sperm and stimulates capacitation in vitro. Seminal plasma alters the ability of epididymal sperm to bind heparin, and several heparin-binding proteins (HBPs) have been identified in bull seminal plasma. This study had three objectives: 1) to identify production sites of seminal plasma HBPs, 2) to determine which HBPs bound to cauda epididymal sperm, and 3) to determine whether presence of HBPs was testosterone dependent. Proteins from bull or rat seminal vesicles, prostates, and bulbourethral glands were separated by heparin affinity high-performance liquid chromatography. HBPs were found in all accessory glands of rats and bulls, but the major source of bovine seminal plasma HBPs appeared to be seminal vesicles. Between 25% and 50% of the protein from each gland bound to the heparin column, and NaCl concentrations required to elute proteins ranged from 0.15 to 1.4 M. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that major HBPs were relatively small, with molecular weights between 13 and 31 kDa, but some HBPs also exhibited higher molecular weights, between 40 and 100 kDa. Radioiodinated HBPs from each bovine gland were incubated with epididymal sperm. Labeled HBPs binding to sperm exhibited molecular weights of 14, 16, 24, and 30 kDa as determined by SDS-PAGE and autoradiography. The HBP content of the accessory sex glands decreased significantly in castrated rats and was restored to levels of sham-operated controls by testosterone replacement. Heparin-binding proteins may play a role in fertilization by attaching to sperm surfaces, enabling heparin-like glycosaminoglycans in the female reproductive tract to induce capacitation.

Role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta 1 in c-myc overexpressing mammary epithelial cells.

We previously showed that TGF alpha synergizes with c-myc in mammary tumorigenesis through inhibition of Myc-induced apoptosis. We therefore examined the effects of growth factors on apoptosis induction in several cell lines from MMTV-myc mammary tumors. When EGF was withdrawn or TGF beta 1 was added, cells became apoptotic after 15 h (by ELISA and morphology). Northern and Western analysis revealed high levels of Bax and p53, and low or undetectable levels of Bcl-2 and Bcl-xS under all treatment condition. In contrast, Bcl-xL expression was highest in the presence of EGF or TGF alpha, with a significant reduction upon removal of EGF or exposure to TGF beta. In mouse mammary tumors, the relative Bcl-xL/Bax ratio was higher in TGF alpha/Myc double transgenics than in Myc single transgenics, in agreement with the in vitro data. Our results suggest a role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta in mammary epithelial cells.

C-myc amplification in breast cancer: a meta-analysis of its occurrence and prognostic relevance.

Data from basic research suggests that amplification of the proto-oncogene c-myc is important in breast cancer pathogenesis, but its frequency of amplification and prognostic relevance in human studies have been inconsistent. In an effort to clarify the clinical significance of c-myc amplification in breast cancer, we conducted a comprehensive literature search and a meta-analysis in which 29 studies were evaluated. The weighted average frequency of c-myc amplification in breast tumours was 15.7% (95% CI = 12.5-18.8%), although estimates in individual studies exhibited significant heterogeneity, P<0.0001. C-myc amplification exhibited significant but weak associations with tumour grade (RR = 1.61), lymph-node metastasis (RR = 1.24), negative progesterone receptor status (RR = 1.27), and postmenopausal status (RR = 0.82). Amplification was significantly associated with risk of relapse and death, with pooled estimates RR = 2.05 (95% CI = 1.51-2.78) and RR = 1.74 (95% CI = 1.27-2.39), respectively. This effect did not appear to be merely a surrogate for other prognostic factors. These results suggest that c-myc amplification is relatively common in breast cancer and may provide independent prognostic information. More rigorous studies with consistent methodology are required to validate this association, and to investigate its potential as a molecular predictor of specific therapy response.

Mapping of the Mod-1 locus on mouse chromosome 9.

A new method for typing the Mod-1 locus on mouse Chromosome (Chr) 9 was developed, based on restriction fragment length polymorphism (RFLP) within a polymerase chain reaction (PCR)-amplified fragment. The new method led us to revise the strain distribution pattern (SDP) of Mod-1 in the BXD (C57BL/6J x DBA/2J) and AKXD (AKR/J x DBA/2J) recombinant inbred (RI) strains. The new SDP eliminates several previously reported examples of double recombination events between Mod-1 and the closest flanking loci in the BXD and AKXD strains. In the BXD strains, the revised SDP of Mod-1 was identical to that of the Mod-1-related D9Rtil locus. Thus, the identity of D9Rtil as a Mod-1-related locus rather than Mod-1 itself is in question. The method was also applied to an interspecific backcross panel between an inbred strain of Mus musculus molossinus (MSM/Ms) and C57BL/6J to map Mod-1 with respect to surrounding microsatellite loci, defining the proximal localization of Mod-1 with respect to D9Mit10 with a genetic distance of 0.6 +/- 0.6 cM.