BRCA1 and BRCA2 mRNA levels are coordinately elevated in human breast cancer cells in response to estrogen

The steady state levels of BRCA1 and BRCA2 mRNAs were shown to be coordinately elevated by the steroid hormone estrogen but not progesterone in the human breast cancer cell lines BT-483 and MCF-7. Two different antiestrogens, trans 4'-hydroxytamoxifen and ICI 182,780, blocked the elevation of BRCA1 and BRCA2 mRNA levels, confirming that the effect was mediated through the estrogen receptor. In BT-483 cells, BRCA1 and BRCA2 mRNA levels were both elevated 18 to 24 h after estrogen stimulation, suggesting that the effect of estrogen was indirect. Cycloheximide blocked the estrogen effect implying that estrogen induces synthesis of an unidentified estrogen-responsive protein(s) that then result in the elevation of BRCA1 and BRCA2 mRNAs.     

The BRCA1 and BRCA2 breast cancer genes

This review discusses the need for sleep, effects of sleep deprivation on behaviour and performance in the military, and sleep management recommendations to optimise combat effectiveness. Most people, regardless of sex or race, prefer 7 to 8 hours of sleep each night. Sleeping during the day is less recuperative. Continuous sleep is more effective than multiple short naps-even when the total hours for naps is more. Ten to 20 minute naps are useful when continuous sleep is not possible. Sleep inertia is the 5 to 30 minute period of sluggishness after awakening and important military tasks should be avoided. Previously, continuous work episodes (CWEs) duration was restricted by limited night vision, unreliable equipment and reduced endurance of military personnel. With improved technology, CWEs are now restricted primarily by endurance which is affected by sleep deprivation. This was one of the experiences noted in recent conflicts (e.g. Desert Storm) by personnel in the air force, army and navy. Since there will be changes in operational requirements, several work-rest-sleep plans must be prepared. Sleeping the preferred 7 to 8 hours per 24 hours the week before an operation may help prepare for optimal performance. Personnel should be familiarised with conditions under which they may sleep. During combat, sleep management should ideally avoid situations where all personnel are exhausted at the same time. As sleep debt accumulates, a person's mood, motivation, attention, alertness, short-term memory, ability to complete routines, task performance (errors of omission more than errors of commission) and physical performance will become more negatively affected. Counter measures must then be taken (e.g. time for sleep or naps, changing routines or rotating jobs). Drugs like caffeine and amphetamine can help personnel stay awake. However, they may also keep them awake when they need to sleep- and on awakening, they could suffer from "hang-overs" and are less efficient. Sleep lost need not be replaced hour-for-hour. Therefore, after operations, personnel need continuous sleep for only 10 to 12 hours as longer sleep increases sleep inertia and delays getting back to normal schedules.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Differential contributions of BRCA1 and BRCA2 to early-onset breast cancer

BACKGROUND: Germ-line mutations in the BRCA1 and BRCA2 genes predispose women to breast cancer. BRCA1 mutations are found in approximately 12 percent of women with breast cancer of early onset, and the specific mutation causing a deletion of adenine and guanine (185delAG), which is present in 1 percent of the Ashkenazi Jewish population, contributes to 21 percent of breast cancers among young Jewish women. The contribution of BRCA2 mutations to breast cancer of early onset is unknown. METHODS: Lymphocyte specimens from 73 women with breast cancer diagnosed by the age of 32 were studied for heterozygous mutations of BRCA2 by a complementary-DNA-based protein-truncation assay, followed by automated nucleotide sequencing. In addition, specimens from 39 Jewish women with breast cancer diagnosed by the age of 40 were tested for specific mutations by an allele-specific polymerase chain reaction. RESULTS: Definite BRCA2 mutations were found in 2 of the 73 women with early-onset breast cancer (2.7 percent; 95 percent confidence interval, 0.4 to 9.6 percent), suggesting that BRCA2 is associated with fewer cases than BRCA1 (P=0.03). The specific BRCA2 mutation causing a deletion of thymine (6174delT), which is found in 1.3 percent of the Ashkenazi Jewish population, was observed in 1 of the 39 young Jewish women with breast cancer (2.6 percent; 95 percent confidence interval, 0.09 to 13.5 percent), indicating that it has a small role as a risk factor for early-onset breast cancer. Among young women with breast cancer, there are BRCA2 mutations that cause truncation of the extreme C terminus of the protein and that may be functionally silent, along with definite truncating mutations. CONCLUSIONS: Germ-line mutations in BRCA2 contribute to fewer cases of breast cancer among young women than do mutations in BRCA1. Carriers of BRCA2 mutations may have a smaller increase in the risk of early-onset breast cancer.     

Moderate frequency of BRCA1 and BRCA2 germ-line mutations in Scandinavian familial breast cancer

Previous studies of high-risk breast cancer families have proposed that two major breast cancer-susceptibility genes, BRCA1 and BRCA2, may account for at least two-thirds of all hereditary breast cancer. We have screened index cases from 106 Scandinavian (mainly southern Swedish) breast cancer and breast-ovarian cancer families for germ-line mutations in all coding exons of the BRCA1 and BRCA2 genes, using the protein-truncation test, SSCP analysis, or direct sequencing. A total of 24 families exhibited 11 different BRCA1 mutations, whereas 11 different BRCA2 mutations were detected in 12 families, of which 3 contained cases of male breast cancer. One BRCA2 mutation, 4486delG, was found in two families of the present study and, in a separate study, also in breast tumors from three unrelated males with unknown family history, suggesting that at least one BRCA2 founder mutation exists in the Scandinavian population. We report 1 novel BRCA1 mutation, eight additional cases of 4 BRCA1 mutations described elsewhere, and 11 novel BRCA2 mutations (9 frameshift deletions and 2 nonsense mutations), of which all are predicted to cause premature truncation of the translated products. The relatively low frequency of BRCA1 and BRCA2 mutations in the present study could be explained by insufficient screening sensitivity to the location of mutations in uncharacterized regulatory regions, the analysis of phenocopies, or, most likely, within predisposed families, additional uncharacterized BRCA genes.     

Androgens down-regulate bcl-2 protooncogene expression in ZR-75-1 human breast cancer cells

Although a large proportion of primary human breast cancers express the androgen receptor, and treatment with androgens exerts beneficial effects in women with breast cancer, the role and especially the mechanism of action of androgens in breast cancer development and growth are not well understood. The potential effect of androgens on bcl-2 protooncogene expression was investigated in a human breast cancer cell line whose proliferation is known to be inhibited by androgens. The estrogen-responsive ZR-75-1 cells were grown in the presence or absence of 5alpha-dihydrotestosterone (DHT), alone or in combination with 17beta-estradiol. DHT caused a marked down-regulation of Bcl-2 protein and messenger RNA levels in both the presence and absence of 17beta-estradiol. The inhibitory effect of DHT was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide. The present data indicate that androgens can down-regulate bcl-2 protooncogene levels via an androgen receptor-mediated mechanism, thus providing a novel mechanism for their known inhibitory effect on breast cancer cell growth.     

Mutations in BRCA1 and BRCA2 in breast cancer families: are there more breast cancer-susceptibility genes?

To estimate the proportion of breast cancer families due to BRCA1 or BRCA2, we performed mutation screening of the entire coding regions of both genes supplemented with linkage analysis of 31 families, 8 containing male breast cancers and 23 site-specific female breast cancer. A combination of protein-truncation test and SSCP or heteroduplex analyses was used for mutation screening complemented, where possible, by the analysis of expression level of BRCA1 and BRCA2 alleles. Six of the eight families with male breast cancer revealed frameshift mutations, two in BRCA1 and four in BRCA2. Although most families with female site-specific breast cancers were thought to be due to mutations in either BRCA1 or BRCA2, we identified only eight mutations in our series of 23 site-specific female breast cancer families (34%), four in BRCA1 and four in BRCA2. According to the posterior probabilities calculated for mutation-negative families, based on linkage data and mutation screening results, we would expect 8-10 site-specific female breast cancer families of our series to be due to neither BRCA1 nor BRCA2. Thus, our results suggest the existence of at least one more major breast cancer-susceptibility gene.     

BRCA1 and BRCA2 in breast cancer families from Wales: moderate mutation frequency and two recurrent mutations in BRCA1

Mutations in the BRCA1/BRCA2 genes account for varying proportions of breast cancer families studied, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations in 17 families from Wales with two or more cases of breast cancer under age 50 and/or ovarian cancer. Eight out of 17 (47%) families had demonstrable mutations. Six out of 17 (35%) carried BRCA1 mutations and 2 out of 17 (12%) carried BRCA2 mutations. Two recurrent mutations in BRCA1 were identified, which appear to represent founder mutations in this population. These data support the existence of additional breast and ovarian cancer susceptibility genes.     

Germline mutations of the BRCA1 and BRCA2 genes in a breast and ovarian cancer patient

Nitric oxide (NO) has been implicated as a modulator of the vascular effects of angiotensin II (ANG II) in the kidney. We used a NO-sensitive microelectrode to study the effect of ANG II on NO release, and to determine the effect of selective inhibition of the ANG II subtype I receptor (AT1) with losartan (LOS) and candesartan (CAN). NO release from isolated and perfused renal resistance arteries was measured with a porphyrin-electroplated, carbon fiber. The vessels were microdissected from isolated perfused rat kidneys and perfused at constant flow and pressure in vitro. The NO-electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. ANG II stimulated NO release in a dose-dependent fashion: 0.1 nM, 10 nM and 1000 nM ANG II increased NO-oxidation current by 85+/-18 pA (n = 11), 148+/-22 pA (n = 11), and 193+/-29 pA (n = 11), respectively. These currents correspond to changes in effluent NO concentration of 3.4+/-0.5 nM, 6.1+/-1.1 nM, and 8.2+/-1.3 nM, respectively. Neither LOS (1 muM) nor CAN (1 nM) significantly affected basal NO production, but both AT1-receptor blockers markedly blunted NO release in response to ANG II (10 nM): 77+/-6% inhibition with LOS (n = 8) and 63+/-9% with CAN (n = 8). These results are the first to demonstrate that ANG II stimulates NO release in isolated renal resistance arteries, and that ANG II-induced NO release is blunted by simultaneous AT1-receptor blockade. Our findings suggest that endothelium-dependent modulation of ANG II-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1-receptor-dependent NO release.     

Effect of smoking on breast cancer in carriers of mutant BRCA1 or BRCA2 genes

BACKGROUND: Smoking has carcinogenic effects, and possibly antiestrogenic effects as well, but it has not been found to be a risk factor for breast cancer in women in the general population. However, hereditary breast cancer is primarily a disease of premenopausal women, and interactions between genes and hormonal and environmental risk factors may be particularly important in this subgroup. METHODS: We conducted a matched case-control study of breast cancer among women who have been identified to be carriers of a deleterious mutation in either the BRCA1 or the BRCA2 gene. These women were assessed for genetic risk at one of several genetic counseling programs for cancer in North America. Information about lifetime smoking history was derived from a questionnaire routinely administered to women who were found to carry a mutation in either gene. Smoking histories of case subjects with breast cancer and age-matched healthy control subjects were compared. Odds ratios for developing breast cancer were determined for smokers versus nonsmokers by use of conditional logistic regression for matched sets after adjustment for other known risk factors. RESULTS: Subjects with BRCA1 or BRCA2 gene mutations and breast cancer were significantly more likely to have been nonsmokers than were subjects with mutations and without breast cancer (two-sided P = .007). In a multivariate analysis, subjects with BRCA1 or BRCA2 mutations who had smoked cigarettes for more than 4 pack-years (i.e., number of packs per day multiplied by the number of years of smoking) were found to have a lower breast cancer risk (odds ratio = 0.46, 95% confidence interval = 0.27-0.80; two-sided P = .006) than subjects with mutations who never smoked. CONCLUSIONS: This study raises the possibility that smoking reduces the risk of breast cancer in carriers of BRCA1 or BRCA2 gene mutations.     

Double heterozygosity for mutations in the BRCA1 and BRCA2 genes in a breast cancer patient

BACKGROUND: Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 confer substantial increased lifetime risk for breast cancer, and in the case of BRCA1, for ovarian carcinoma as well. These two genes alone account for the vast majority of hereditary breast cancer families. Numerous mutations have been described in each gene, the majority of which are small insertions or deletions resulting in expression of a truncated protein. MATERIALS AND METHODS: Several common mutations can be detected using a polymerase chain reaction-mediated, site-directed mutagenesis assay, which transforms the amplicon derived from either the wild-type or mutant allele by adding or removing a restriction endonuclease site. We screened 49 putative sporadic breast tumors using this methodology, targeting four BRCA1 mutations (185delAG, 5382insC, R1443X, and E1250X) and a single BRCA2 mutation (6174delT). RESULTS: Using the polymerase chain reaction-mediated, site-directed mutagenesis assay, we identified two mutations, namely, a 185delAG mutation (BRCA1) and a 6174delT mutation (BRCA2). Interestingly, these two mutations were found in the same sample. None of the remaining 48 breast tumors showed evidence of these mutations. Allele-specific oligonucleotide probes were then employed in conjunction with the Universal GeneComb Test Kit, which confirmed the presence of mutations. CONCLUSIONS: Our data suggest that the common germline BRCA1 and BRCA2 mutations are infrequently encountered in sporadic breast cancers. The one case with dual BRCA1 and BRCA2 mutations suggests that this tumor may be hereditary in origin, despite the lack of a positive family history. Double heterozygosity for mutations in BRCA1 and BRCA2 may have increasingly significant implications with regard to predisposition to breast cancer.     

Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and beta-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNKI or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicin-induced JNKI activity, but had no effect on JNKI activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs.     

Effect of BRCA1 and BRCA2 on the association between breast cancer risk and family history

BACKGROUND: The discovery of BRCA1 and BRCA2 has led to a reassessment of the association between family history of breast/ovarian cancer and breast cancer risk after controlling for carrier status for mutations in the BRCA1 and BRCA2 genes. We examined whether family history of breast cancer remains a predictive risk factor for this disease after carrier status for BRCA1 and/or BRCA2 mutations is taken into consideration. METHODS: The data are from 4730 case subjects with breast cancer and 4688 control subjects enrolled in the Cancer and Steroid Hormone Study. The probability of being a BRCA1 and/or BRCA2 gene carrier was calculated for each woman. Among predicted noncarriers, logistic regression was used to assess the relationship (odds ratios and 95% confidence intervals [CIs]) between case or control status and family history of breast or ovarian cancer. Estimates of age-specific breast cancer risk are presented by predicted carrier status. RESULTS: Among predicted noncarriers, case subjects were 2.06 times (95% CI = 1.69-2.50) and 1.24 times (95% CI = 1.17-1.32) more likely to report a first-degree or second-degree family history of breast cancer, respectively, than were control subjects. Case subjects were 1.99 times (95% CI = 1.63-2.44), 1.66 times (95% CI = 1.18-2.38), and 2.23 times (95% CI = 0.21-24.65) more likely to report an affected mother, sister, or both, respectively, than were control subjects. A family history of ovarian cancer was not statistically significantly associated with breast cancer risk. Noncarriers were predicted to have a lifetime risk of 9% of developing breast cancer compared with a 63% risk for carriers. CONCLUSIONS: Among women with a moderate family history of breast cancer, i.e., predicted noncarriers of BRCA1 and/or BRCA2 mutations, family history remains a factor in predicting breast cancer risk. In families with breast and ovarian cancers, the aggregation of these two cancers appears to be explained by BRCA1/BRCA2 mutation-carrier probability.     

Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.     

Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells

A series of N-substituted 4-amino-2,2-diphenylbutyramide derivatives was prepared as part of a search for subtype-selective antimuscarinic agents. The representative compound KRP-197, bearing a 2-methylimidazole ring as a surrogate of aliphatic amine, was found to be a highly potent and both M1- and M3-selective antimuscarinic agent.     

BRCA2 germline mutations in Swedish breast cancer families

Life-threatening situations in sarcoidosis are extremely rare. They may be due to failure of vital organs--lungs, heart, kidney, liver and brain--and usually due to irreversible fibrosis. Respiratory failure follows irreversible pulmonary fibrosis and the development of cor pulmonale. Cardiac sarcoidosis is more sinister for it may be silent, ill-recognised with sudden death or high morbidity. It needs sophisticated techniques to uncover this latent iceberg. Renal failure may be due to granulomatous interstitial nephritis and/or nephrocalcinosis. Hepatic failure is due to intrahepatic cholestasis, portal hypertension and bleeding oesophageal varices. Neurosarcoidosis carries a mortality of 10 per cent, over twice that of sarcoidosis overall. The treatment of each situation is discussed including organ transplantation.