Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells.

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.

Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables such as cabbage, broccoli, and Brussels sprouts, has been shown to reduce the incidence of spontaneous and carcinogen-induced mammary tumors. Treatment of cultured human MCF7 breast cancer cells with I3C reversibly suppresses the incorporation of [3H]thymidine without affecting cell viability or estrogen receptor (ER) responsiveness. Flow cytometry of propidium iodide-stained cells revealed that I3C induces a G1 cell cycle arrest. Concurrent with the I3C-induced growth inhibition, Northern blot and Western blot analyses demonstrated that I3C selectively abolished the expression of cyclin-dependent kinase 6 (CDK6) in a dose- and time-dependent manner. Furthermore, I3C inhibited the endogenous retinoblastoma protein phosphorylation and CDK6 phosphorylation of retinoblastoma in vitro to the same extent. After the MCF7 cells reached their maximal growth arrest, the levels of the p21 and p27 CDK inhibitors increased by 50%. The antiestrogen tamoxifen also suppressed MCF7 cell DNA synthesis but had no effect on CDK6 expression, while a combination of I3C and tamoxifen inhibited MCF7 cell growth more stringently than either agent alone. The I3C-mediated cell cycle arrest and repression of CDK6 production were also observed in estrogen receptor-deficient MDA-MB-231 human breast cancer cells, which demonstrates that this indole can suppress the growth of mammary tumor cells independent of estrogen receptor signaling. Thus, our observations have uncovered a previously undefined antiproliferative pathway for I3C that implicates CDK6 as a target for cell cycle control in human breast cancer cells. Moreover, our results establish for the first time that CDK6 gene expression can be inhibited in response to an extracellular antiproliferative signal.

National Delphi study to determine competencies for nursing leadership in public health.

PURPOSE: To identify competencies needed by nurse leaders in public health programs. DESIGN: Five-round national Delphi. SAMPLE: Convenience sample of members of major public health nursing associations and nurse and non-nurse public health leaders in the USA. METHODS: Mailed survey in 1994-1995 using a modified snowball technique based on a modification of the Pew Foundation health professions' competencies for Round 1. Four additional rounds produced consensus. FINDINGS: Initially, 62 competencies were identified. Factor analysis resulted in four factors: political competencies, business acumen, program leadership, and management capabilities; 57 competencies were clustered in the four groupings and accounted for 91.4% of the variance. CONCLUSIONS: Graduate schools in nursing and public health must prepare students with broad-based competencies from a variety of disciplines. Findings of this national survey provide a database for curriculum development and evaluation of programs to prepare nurse leaders for roles in public health-based delivery systems.

Observation evaluation to assess race and educational bias in state-mandated standard testing of nurse aides in nursing homes.

This article presents an assessment of whether race, education, gender, or other testing bias was present in a state-mandated nurse aide competency test. This assessment was carried out with data from two sources: (a) a statewide standardized test for all nurse aides that was given by a nationally known testing company, (b) an independent observational evaluation with a Behaviorally Anchored Rating Scale (BARS) for nurse aides' performance that was carried out by the investigators. The results show that race and education level were predictors of performance on written and manual portions of the standardized test. Gender, age, and years of experience were also shown to predict test success. Comparing data from the two sources suggests that a possible bias in the standardized nurse aid test. The independent observation of performance on the job with the BARS is shown to be less biased.

Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging.

Antioxidants may play an important role in preventing free radical damage associated with aging by interfering directly in the generation of radicals or by scavenging them. We investigated the effects of a high vitamin E and/or a high beta-carotene diet on aging of the anion transporter, band 3, in lymphocytes and brain. The band 3 proteins function as anion transporters, acid base regulators, C02 transporters, and structural proteins that provide a framework for membrane lipids and that link the plasma membrane to the cytoskeleton. Senescent cell antigen (SCA), which terminates the life of cells, is a degradation product of band 3. This study was conducted as a double-blind study in which eight groups of middle-aged or old mice received either high levels of beta-carotene and/or vitamin E or standard levels of these supplements in their diets. Anion transport kinetic assays were performed on isolated splenic lymphocytes. Immunoreactivity of an antibody that recognizes aging changes in old band 3 preceding generation of SCA was used to quantitate aged band 3 in brain tissue. Results indicate that vitamin E prevented the observed age-related decline in anion transport by lymphocytes and the generation of aged band 3 leading to SCA formation. beta-Carotene had no significant effect on the results of either assay. Since increased aged band 3 and decreased anion transport are initial steps in band 3 aging, which culminates in the generation of SCA and cellular removal, vitamin E prevents or delays aging of band 3-related proteins in lymphocytes and brain.

Posttranslational changes in band 3 in adult and aging brain following treatment with ergoloid mesylates, comparison to changes observed in Alzheimer's disease.

Band 3, the most heavily used anion transport system in vertebrates, ages as cells and tissues age. Posttranslational changes in band 3 in adult and aging brain were investigated following treatment with ergoloid mesylates and compared to changes observed in Alzheimer's disease (AD). The study was conducted in a double blind fashion and was decoded only after the study was completed. The following posttranslational changes in brain band 3 occur with age: increased breakdown of band 3; decreased phosphorylation; and decreased anion transport. Autoantibodies to senescent cell antigen (SCA) synthetic peptides residue 538-554 and 812-827 increase with age, but antibodies to the former peptide are significantly reduced in ergoloid mesylate treated old mice. This is a critical transport region of band 3. Results showed the aged/altered band 3 increased in Alzheimer's disease (AD) as determined by quantitative antibody binding. Ergoloid mesylates altered the age-related posttranslational changes as follows: the observed age-related decrease in brain band 3 was partially reversed and anion transport was increased. This is consistent with the data indicating decreased autoantibodies to a critical anion transport segment of band 3. Aging appears to result in damage to a critical transport region of the anion transporter which is reflected by decreased anion transport, increased breakdown, alteration of the molecule itself, and an increase in autoantibodies to the region. Ergoloid mesylates seem to protect against this damage.

Band 3, the anion transporter, is conserved during evolution: implications for aging and vertebrate evolution.

A cDNA fragment corresponding to a highly evolutionarily conserved region of the major anion transport protein band 3 was cloned from lamprey mRNA using PCR homology probing. This is the first report providing evidence for a band-3 like transporter in the lowest vertebrates, the agnathostomes. Semi-quantitative PCR showed expression similar to that of higher vertebrates. Lamprey serum contains antibody-like molecules that bind to synthetic peptides of band 3 comprising senescent cell antigen, an aging antigen that terminates the life of cells. The high degree of homology found in nucleic acid and derived proteins sequence and the reaction of "antibodies" in lamprey serum with senescent cell antigen peptides of band 3 suggests that lamprey band 3 plays a role comparable to that in higher vertebrates.

Molecular variants in the P450c11AS gene as determinants of aldosterone synthase activity in the Dahl rat model of hypertension.

Dahl salt-sensitive (S) and salt-resistant (R) rats are widely used to study genetic determinants of salt-sensitive hypertension. Differences in blood pressure under a high sodium diet in these two strains may be due to differences in the synthesis of 18-OH-11-deoxycorticosterone (18-OH DOC). This difference in 18-OH-DOC synthesis is due to mutations in the Dahl R rat's gene for P450c11 beta (11 beta-hydroxylase), an adrenal enzyme involved in the synthesis of both corticosterone and 18-OH DOC from 11-deoxycorticosterone. Aldosterone/renin ratios in plasma and in the adrenals are greater in Dahl S than R rats, suggesting an altered physiologic relationship between the renin-angiotensin and aldosterone systems between these strains. We demonstrate that the mRNA for P450c11AS, (aldosterone synthase), an enzyme required for aldosterone synthesis, is identical in the Dahl S rat and in normotensive Sprague-Dawley rats, but that P450c11AS mRNA from the Dahl R rat contains 7 mutations that result in two amino acid substitutions. These two changes result in a form of P450c11AS that has a greater apparent Vmax and lower apparent Km, resulting in an enzyme that catalyzes the conversion of 11-deoxycorticosterone to aldosterone at a greater rate in Dahl R rats than the P450c11AS in Dahl S rats or Sprague-Dawley rats. Although plasma and adrenal renin are lower in Dahl S versus R rats, the regulation of P450c11AS mRNA expression in rats fed a low and high salt diet are identical in these strains. The current findings may explain both the reduced aldosterone concentrations and increased aldosterone/renin ratios previously reported in the Dahl S versus Dahl R rat.

Rodent models for studying steroids and hypertension: from fetal development to cells in culture.

We have used several different approaches to study the role of steroids in hypertension, including rodent in vivo models, transgenic animals, and cell culture systems. Using the developing rodent fetus as a model for the ontogeny of regulation of glucocorticoid and mineralocorticoid synthesis, we found that in the developing rodent fetus, expression of both P450scc (cholesterol side chain cleavage) and P450c11 beta (11 beta-hydroxylase) mRNAs occur early, before there is complete organization of the fetal adrenal. Even after the zones of the adrenal are evident, the fetal adrenal still does not express the glomerulosa-specific P450c11AS (aldosterone synthase) mRNA. Stimulating maternal adrenal mineralocorticoid or glucocorticoid synthesis does not affect accumulation of fetal adrenal steroidogenic mRNAs, suggesting that the rodent fetal adrenal may be somewhat transcriptionally quiescent in vivo. We also used two different transgenic rodent systems to study the roles of steroids in hypertension. Using promoter-directed tumorigenesis in transgenic mice, we created transgenic mice that expressed SV40 T antigen under control of the P450scc promoter. Massive adrenal tumors, but not gonadal tumors, developed in all transgenic mice, and cells from these tumors were easily cultured. Using a novel selection tactic, we obtained several adrenocortical cell lines which have distinct characteristics, suggesting they were locked into various stages of differentiation; both expression of steroidogenic mRNAs and the steroids synthesized differ among the lines. Regulation of steroid synthesis and mRNA abundance also varies among cell lines. Several cell lines also express mouse renin, and its synthesis, secretion, and mRNA abundance is also hormonally regulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Band 3 and its peptides during aging, radiation exposure, and Alzheimer's disease: alterations and self-recognition.

An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding. Band 3 is a ubiquitous membrane transport protein found in the plasma membrane of diverse cell types and tissues, and in nuclear, mitochondrial, and golgi membranes. Band 3 in tissues such as brain performs the same functions as it does in red blood cells forming senescent cell antigen. Oxidation is a mechanism for generating senescent cell antigen. The aging antigenic sites reside on human band 3 map residues 538-554, and 812-830. Carbohydrate moieties are not required for the antigenicity or recognition of senescent cell antigen. Anion transport site were mapped to residues 588-594, 822-839, and 869-883. The aging vulnerable site which triggers the antigenic site and the transport sites of band 3 were mapped using overlapping synthetic peptides along the molecule. Naturally occurring autoantibodies to regions of band 3 comprising both senescent cell antigen and B cells producing these antibodies were demonstrated in the sera of normal, healthy individuals. The presence of these antibodies tend to increase with age. Individuals with autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) have increased antibodies to senescent cell antigen peptides. Radiation exposure results in an increase in antibodies to peptides 588-602 which lies in a transport region containing the aging vulnerable site. Band 3 ages as cells and tissues age. Our studies, to date, indicate, that the anion transport ability of band 3 decreases in brains and lymphocytes from old mice. This decreased transport ability precedes obvious structural changes such as band 3 degradation and generation of SCA, and is the earliest change thus far detected in band 3 function. Other changes include a decreased efficiency of anion transport (decreased Vmax) in spite of an increase in number of anion binding sites (increased Km), decreased glucose transport, increased phosphorylation, increased degradation to smaller fragments as detected by quantitative binding of antibodies to band 3 breakdown products and residue 812-830, and binding of physiologic IgG autoantibodies in situ. The latter 3 findings indicate that post-translational changes occur. In Alzheimer's Disease (AD), our results indicate that post-translational changes occur in band 3. These include decreased band 3 phosphorylation of a 25-28kD segment, increased degradation of band 3, alterations in band 3 recognized by antibodies, and decreased anion and glucose transport by blood cells. Serum autoantibodies were increased in AD patients compared to controls to band 3 peptide 822-839. This band 3 residue lies in an anion transport/binding region.

Ear tag induced Staphylococcus infection in mice.

Mice used in a 2-year oral toxicity study developed a progressive, moist dermatitis. The initial lesions were seen around the ears in which metal identification tags had been placed and usually progressed to include the skin of the neck and shoulder. Clinically, the mice were pruritic, lost weight, had rough coats, and became moribund. The predominant finding at necropsy was pale brown kidneys with irregular granular surfaces. Histologically, there was inflammation and focal-to-diffuse necrosis in the visceral organs and affected skin. The predominant organism isolated from the skin, kidneys and heart blood was Staphylococcus aureus. This bacterium is a common inhabitant of the skin of conventionally housed mice and its isolation from the kidneys and blood suggested that the portal of entry was the wound caused by the insertion of the metal ear tag.

Spontaneous disseminated cytomegalic inclusion disease in an ageing laboratory mouse.

An untreated C57BL/6NCrlBR male mouse in a life-long longevity study, killed in extremis at 761 days of age, had mild focal necrotic hepatitis and extensive focal necrotic adrenalitis. Characteristic cytomegalic cells bearing intranuclear and cytoplasmic inclusions and cytoplasmic vacuoles were seen in necrotic areas. Scattered intranuclear inclusions were also seen in the reticulum cells of the spleen and acinar epithelium of the submaxillary glands. We believe this to be the first report of spontaneous disseminated cytomegalic inclusion disease in aged mice.