Clinician satisfaction with vaccination visits and the role of multiple injections, results from the COVISE Study (Combination Vaccines Impact on Satisfaction and Epidemiology).

Because little is known about clinician satisfaction with infant vaccination visits, we measured satistaction and the effects of the number of injections on satisfaction. Clinicians from 35 pediatric centers self-administered a questionnaire using visual analog scales augmented by a Likert scale. All 95 pediatricians and 137 nonphysician vaccinators responded. In both populations, increased injections predicted decreased overall satisfaction, and decreased satisfaction with obtaining consent, time to prepare/administer, getting upset during administration, and time to update records (each p<0.01). Satisfaction decreased markedly, on each measure, at 4-injection visits, 5-injection visits, or both.

A hypersensitive estrogen receptor-alpha mutation in premalignant breast lesions.

The best current model of breast cancer evolution suggests that most cancers arise from certain premalignant lesions. We have identified a common (34%) somatic mutation in the estrogen receptor (ER)-alpha gene in a series of 59 typical hyperplasias, a type of early premalignant breast lesion. The mutation, which affects the border of the hinge and hormone binding domains of ER-alpha, showed increased sensitivity to estrogen as compared with wild-type ER-alpha in stably transfected breast cancer cells, including markedly increased proliferation at subphysiological levels of estrogen. The mutated ER-alpha exhibits enhanced binding to the TIF-2 coactivator at low levels of hormone, which may partially explain its increased estrogen responsiveness. These data suggest that this mutation may promote or accelerate the development of cancer from premalignant breast lesions.

Auto-regulation of the estrogen receptor promoter.

The presence or absence of estrogen receptor (ER) plays a key role in the diagnosis and treatment of breast tumors. It is known that patients with breast tumors classified as ER-positive have a better prognosis. Observations such as this have led us to explore the question of what makes some breast tumors overexpress ER whereas others express either very low levels or none at all. To begin a study of ER regulation, we first chose to examine a 200 bp region of the ER promoter located immediately upstream from the transcribed sequence of the human ER gene. We found that this region of the ER promoter contained basal activity when transiently transfected into ER-negative HeLa cells. ER promoter activity was further increased by co-transfection of a wild-type ER expression vector, and this increased activity was hormone-dependent. Several ER deletion mutant constructs were also able to increase the activity of the ER promoter fragment, but none could support equivalent activity as was seen with the full-length ER. Therefore, we conclude that the ER can contribute to its own expression, and we hypothesize that this auto-regulation may contribute to its overexpression in some breast tumors.

Differential expression of wild-type and variant ER mRNAs by stocks of MCF-7 breast cancer cells may account for differences in estrogen responsiveness.

The MCF-7 human breast cancer cell line is routinely used as a model system for the study of ER+, estrogen-responsive human breast cancer. Although most investigators have found these cells to be estrogen-responsive, there are reports that some stocks of MCF-7 cells may be either insensitive or may display decreased sensitivity to the mitogenic effects of estrogen. We report here that differences in estrogen responsiveness appear to be related to varying ratios of wild type to variant ER mRNAs. MCF-7 stocks which express a high ratio of wild type to variant ER transcripts are more responsive to the mitogenic effects of physiological concentrations of estradiol than stocks which express an elevated ratio of exon 5 deletion variant to wild type ER transcripts.

Coexpression of wild-type and variant oestrogen receptor mRNAs in a panel of human breast cancer cell lines.

Wild-type as well as variant oestrogen receptor (ER) mRNAs with exon 5 and 7 deleted were identified in a panel of human breast tumour cell lines by reverse transcriptase-polymerase chain reaction followed by dideoxynucleotide sequence analysis, and then quantitated by ribonuclease protection analysis. All cell lines categorised as ER+ by ligand-binding analysis expressed both wild-type and variant ER transcripts. Most cell lines classified as ER- did not express any ER transcript. However, three ER- cell lines (BT-20, MDA-MB-330 and T47Dco) expressed both wild-type and variant transcripts. A differential pattern of expression of wild type to variant was seen in both ER+ and ER- cell lines, however this pattern was not paralleled by differences in ligand-binding activity. Breast tumour cell lines previously classified as ER- expressed significantly lower levels of ER transcripts than did their ER+ counterparts. In view of these findings, as well as earlier reports that the exon 5 deletion ER variant encodes a dominant-positive receptor, it seems clear that some cell lines are misclassified as ER-, and express both wild-type and variant ER mRNAs, and that the overexpression of this variant may account, in part, for their oestrogen-independent phenotype.

Expression of a constitutively active estrogen receptor variant in the estrogen receptor-negative BT-20 human breast cancer cell line.

Estrogen receptor (ER) expression by breast tumors is an important predictor of disease-free survival in breast cancer patients and, more importantly, is a strong predictor of response to endocrine therapy. Variant forms of the ER may play an important role in the loss of hormone responsiveness and the progression to hormone independence. We have examined a panel of human breast tumor cell lines, both ER-positive and ER-negative, and have identified an ER mRNA variant containing a deletion of exon 5 in the ER-negative BT-20 and ER-positive MCF-7 cell lines. This exon 5 deletion variant has been previously reported to be overexpressed in ER-negative/progesterone receptor-positive breast tumors. Using RNase protection analysis, we have found that the predominant ER transcript in the BT-20 cells is the exon 5 deletion variant, while the principal transcript in MCF-7 cells is the wild-type ER mRNA. The variant ER transcript is translated into a truncated receptor protein of approximately M(r) 42,000 when expressed in yeast and, more important, in breast tumor cells. This is the first demonstration of an exon 5 deletion variant ER protein. Functional analysis has shown that this variant ER possesses constitutive transcriptional regulatory activity with respect to an estrogen-regulated reporter gene construct in a yeast expression system. The presence of this ER variant in breast tumor cell lines, as well as breast tumor biopsies and uterine tissue, suggests that it is a naturally occurring variant that may arise by alternative splicing, and whose overexpression may be involved in the progression of breast tumors to a hormone-independent state.

Melatonin decreases estrogen receptor expression in the medial preoptic area of inbred (LSH/SsLak) golden hamsters.

Daily late afternoon injections of melatonin (25 micrograms/day s.c.) were found to reduce the number of cells expressing estrogen receptor immunoreactivity in the medial preoptic area of ovariectomized inbred (LSH/SsLak) golden hamsters. Employing immunocytochemical analysis with the H222 monoclonal antibody to the human estrogen receptor, we examined the effects of melatonin on estrogen receptor expression in the hypothalamus, particularly the medial preoptic area, of ovariectomized virgin female hamsters. Analysis of the results showed that melatonin administration induced a 50-70% decrease in numbers of estrogen receptor-immunoreactive neurons in the medial preoptic area of ovariectomized female hamsters. Furthermore, an overall qualitative decrease in the intensity of estrogen receptor immunoreactivity was observed. In intact regularly cycling female hamsters used to monitor the efficacy of melatonin treatment, there were significant reductions in the serum levels of FSH, LH, and prolactin as measured by radioimmunoassay and in uterine and pituitary weights after 8 wk of melatonin treatment. These results suggest that melatonin may exert its anti-reproductive effects in hamsters by modulating estrogen receptor levels in medial preoptic area neurons, thus influencing steroid feedback mechanisms.