EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

Lack of association between tumor necrosis and hsp-27 expression in primary breast cancer

An echocardiographical and clinical pathological investigation of the rapid loss of weight by a yearling thoroughbred filly revealed an atrial septal defect, a ventricular septal defect and hyperfibrinogenaemia. A post mortem examination confirmed the cardiac abnormalities and revealed a severe thoracic aortitis. It is proposed that the idiopathic thoracic aortitis contributed to the horse's compromised cardiovascular homoeostasis.     

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.     

CDK4 down-regulation induced by paclitaxel is associated with G1 arrest in gastric cancer cells

Paclitaxel induces a cell cycle block at G2-M phase by preventing the depolymerization of microtubules and induces p53-independent apoptosis in many cancer cells. We observed that gastric cancer cells treated with paclitaxel have shown a cyclin-dependent kinase (CDK)4 down-regulation. This paclitaxel-induced CDK4 down-regulation resulted in a cell cycle arrest at G1-S phase. To confirm this observation, we prepared stable transfectants that overexpressed CDK4 and analyzed the cell cycle progression. Ectopic expression of CDK4 in SNU cells resulted in a release of paclitaxel-induced G1 arrest. The release of G1 arrest by enforced expression of CDK4 seems to make the cells more sensitive to paclitaxel-induced apoptosis. From this finding, we could then suggest that paclitaxel treatment induces both G1-S and G2-M blocks in the cell cycle progression of gastric cancer cells.     

Cyclin E2, a novel human G1 cyclin and activating partner of CDK2 and CDK3, is induced by viral oncoproteins

G1 cyclin E controls the initiation of DNA synthesis by activating CDK2, and abnormally high levels of cyclin E expression have frequently been observed in human cancers. We have isolated a novel human cyclin, cyclin E2, that contains significant homology to cyclin E. Cyclin E2 specifically interacts with CDK inhibitors of the CIP/KIP family and activates both CDK2 and CDK3. The expression of cyclin E2 mRNA oscillates periodically throughout the cell cycle, peaking at the G1/S transition, and exhibits a pattern of tissue specificity distinct from that of cyclin E1. Cyclin E2 encodes a short lived protein whose turnover is most likely governed by the proteasome pathway and is regulated by phosphorylation on a conserved Thr-392 residue. Expression of the viral E6 oncoprotein in normal human fibroblasts increases the steady state level of cyclin E2, but not cyclin E1, while expression of the E7 oncoprotein upregulates both. These data suggest that the expression of these two G1 E-type cyclins may be similarly regulated by the pRb function, but distinctly by the p53 activity.     

Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.     

Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro

IPRS is a freely available software system which consists of about 250 library functions in C, and a set of application programs. It is designed to run under UNIX and comes with full source code, system manual pages, and a comprehensive user's and programmer's guide. It is intended for use by researchers in human vision, pattern recognition, image processing, machine vision and machine learning.     

Interaction between the MEC1-dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality.     

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells

OBJECTIVES: The aim of this study was to evaluate the tolerance and outcome of elderly cervical carcinoma patients treated with radiation therapy (RT). METHODS: Three hundred ninety-eight patients with stage I-III cervical carcinoma treated with definitive RT were analyzed. Patients were divided into nonelderly (ages 35-69) (n = 338) and elderly (ages >/= 70) (n = 60) groups. A comparison of patient, tumor and treatment factors, morbidity, and outcome was performed. Median follow-up was 81 months. RESULTS: Elderly patients had a higher rate of comorbid conditions including diabetes (P = 0. 02), coronary artery disease (P = 0.003), and hypertension (P = 0. 001) than younger patients. Comorbid conditions in the elderly resulted in more frequent treatment breaks and less ability to undergo definitive treatment with intracavitary RT (ICRT). While the 5-year actuarial disease-free (DFS) and cause-specific (CSS) survival rates were comparable between the two groups, disease recurrence and death from cervical cancer were more common beyond 5 years in the elderly group. When patients not undergoing ICRT and those with treatment protraction were excluded, differences in the DFS and CSS curves were no longer evident. The frequency and severity of acute and chronic sequelae were similar. CONCLUSIONS: Elderly cervical carcinoma patients have an equivalent overall outcome following radiation therapy to younger patients when comparable treatment is delivered. Age per se is not associated with a higher rate of acute or chronic sequelae. Comorbid conditions adversely impacted on the quality and delivery of RT in the elderly group and help explain observed differences in outcome based on age.     

Aberrant expression of cyclin A and cyclin B1 proteins in oral carcinoma

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Aberrant expression of cyclin proteins has been found in a number of human cancers, including carcinomas of the head and neck, where amplification of the cyclin D1 gene is a common finding. The objective of this study was to examine cell cycle kinetics in oral carcinomas by determining the expression of the S phase protein cyclin A and the M phase protein cyclin B1. Routinely processed tissue sections of 50 oral squamous cell carcinomas from the floor of the mouth were stained by immunohistochemistry for cyclin A, cyclin B1 and Ki-67 proteins. Ten specimens of normal epithelium from the floor of the mouth were used as controls. The number of cells showing nuclear staining for cyclin A, cyclin B1 and Ki-67 proteins was determined by computer image analysis. There were 17 well-differentiated, 25 moderately differentiated and 8 poorly differentiated tumours. Mean counts for cyclin A (29.50+/-4.10, mean+/-95% CI), cyclin B1 (2.05+/-0.30) and Ki-67 (49.46+/-5.91) proteins in the carcinomas were significantly higher than counts for the normal epithelial controls (cyclin A: 9.30+/-1.72; cyclin B1: 1.01+/-0.36; Ki-67: 17.40+/-4.17). For cyclin A, cyclin B1 and Ki-67, mean staining scores for all tumour grades were significantly higher than controls. There was a strong correlation between Ki-67 and cyclin A scores in all tumour groups (r2=0.68); however, the correlations between cyclin B1 and cyclin A scores (r2=0.35) and between cyclin B1 and Ki-67 scores (r2= 0.39) were weak. We conclude that there is overexpression of cyclin A and cyclin B1 proteins in oral carcinoma. Furthermore, the poor correlations for cyclin B1 scores with other cell cycle indices suggest that there may be aberrant cell cycle progression at the G2/M checkpoint in oral carcinomas.     

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.     

Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development

Postnatal development of Leydig cells can be divided into three distinct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7alpha-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5alpha-reductase, or E2 (50 nM). Proliferative capacity was measured by assaying [3H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A2 and G1, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), intermediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). Similarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), intermediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A2 mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.14), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G1 mRNA levels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 +/- 0.07), and lowest in PLC (0.12 +/- 0.02). The relative protein levels of cyclin A2 and G1 paralleled their mRNA levels. Increased proliferative capacity was observed in PLC and ILC, but not ALC, after treatment with either LH or IGF-I. Treatment with MENT increased proliferative capacity only in ILC and had no effect in any other group. Treatment with E2 decreased proliferative capacity in PLC but not in ILC or ALC. The changes in proliferative capacity after hormonal treatment paralleled cyclin A2 mRNA and were the inverse of cyclin G1 mRNA levels. We conclude that: 1) decreased cyclin A2 and increased cyclin G1 are associated with the withdrawal of the Leydig cell from the cell cycle; 2) the proliferative capacity of Leydig cells is regulated differentially by hormones and is progressively lost during postnatal differentiation.