A Potential Strategy for Atherosclerosis Prevention in Modernizing China - Hyperhomocysteinemia, MTHFR C677T Polymorphism and Air Pollution (PM2.5) on Atherogenesis in Chinese Adults.

BACKGROUND: Atherosclerosis is one of the most important global health hazards and air pollution (AP, PM2.5) has been implicated. In addition to traditional risk factors hyperhomocysteinemia (HC) has been recognized in many parts of China related to risk of stroke. METHODS: To evaluate the impact of HC (homocysteine >14µmol/l) and PM2.5 air pollution on atherogenesis in modernizing China, we studied 756 asymptomatic Chinese in China from 1998-2007. PM2.5 exposure, HC, folate, and methylenetetrahydrofolate reductase (MTHFR) C/T genotype were evaluated. Brachial flow-mediated dilation (FMD) and carotid intima-media thickness (IMT) were measured by ultrasound. Locations were categorized as zones 1, 2 and 3, with increasing PM2.5 exposure. RESULTS: HC was higher (19.4±13.1 and 27.1±25.1µmol/l) in high PM2.5-polluted zones 2 and 3 than in zone 1 (9.7±4.5µmol/l, p<0.0015). The top HC tertile was characterized by lower folate and vitamin B12, but a higher proportion of the MTHFR TT genotype, Metabolic Syndrome (MS) and PM2.5 level (p=0.0018). FMD was significantly lower (7.3±2.3%) and carotid IMT thicker (0.63±0.12mm) in the top HC tertile, compared with low HC tertile (8.4±2.5%, p<0.0001; 0.57±0.1mm, p<0.0001 respectively). Similar differences in FMD and IMT were seen in zones 2 and 3, compared with zone 1 (p<0.0001). On multivariate regression, HC was related to male gender (beta=0.106, p=0.021), MTHFR-TT (beta=0.935, p<0.0001), locations (beta=0.230, p<0.0001) and folate-MTHFR interaction (beta=-0.566, p<0.0001). FMD was related to age (beta= -0.221; p<0.0001), male gender (beta= -0.194, p=0.001) PM2.5 and location (beta=-0.285 to -0.303, p<0.0001). Carotid IMT was related to PM2.5 (beta=0.173, p<0.0001), HC (0.122, p=0.006) but not to MTHFR or location, independent of age, gender, MS, and LDL-C. No significant HC-PM2.5 interaction effect on FMD and IMT was observed. CONCLUSION: HC and PM2.5 pollution but not MTHFR genotype were both related to carotid IMT, independent of other traditional risk factors. This has potential implications in dietary and AP strategies for atherosclerosis prevention in China.

The impact of particulate matter air pollution (PM2.5) on atherosclerosis in modernizing China: a report from the CATHAY study.

BACKGROUND: Air pollution has been associated with an increase in cardiovascular diseases incidence. To evaluate whether air pollution can accelerate atherogenic processes, we assessed the effects of air pollution on important surrogate markers of atherosclerosis [brachial flow-mediated dilation (FMD) and carotid intima-media thickness (IMT)]. METHODS: A total of 1656 Han Chinese (mean age 46.0 + 11.2 years; male 47%) in Hong Kong, Macau, Pun Yu, Yu County and the 3-Gorges Territories (Yangtze River) were studied between 1996 and 2007 [Chinese Atherosclerosis in the Aged and Young Project (the CATHAY Study)]. Cardiovascular risk profiles were evaluated. Particulate matter with an aerodynamic diameter <2.5 µm (PM2.5) parameters were computed from satellite sensors. Brachial FMD and carotid IMT were measured by ultrasound. RESULTS: Health parameters [age, gender, body mass index, waist : hip ratio (WHR) and glucose)] were similar in lowest and highest PM2.5 exposure tertiles, systolic and diastolic blood pressures and triglycerides were higher (P < 0.001) and low-density cholesterol (LDL-C) was lower in the top PM2.5 tertile (P < 0.001). Brachial FMD [7.84 ± 1.77, 95% confidence interval (CI) 7.59-8.10%, vs 8.50 ± 2.52, 95% CI 8.23-8.77%, P < 0.0001) was significantly lower and carotid IMT (0.68 ± 0.13 mm, 95% CI 0.67-0.69 mm vs 0.63 mm ± 0.15 mm 95% CI 0.62-0.64 mm; P < 0.0001) was significantly thicker in the top PM2.5 tertile compared with the lowest tertile. On multiple regression, FMD was inversely related to PM2.5 (beta = 0.134, P = 0.015) independent of gender, age and blood pressure (model R2 = 0.156, F-value = 7.6, P < 0.0001). Carotid IMT was significantly correlated with PM2.5 exposure (beta = 0.381, P < 0.0001) independent of age, location, gender, WHR, blood pressure and LDL-C (model R2 = 0.408, F-value = 51.4, P-value <0.0001). CONCLUSIONS: Air pollution is strongly associated with markers of early atherosclerosis, suggesting a potential target for preventive intervention.

[Exploratory research on developing lung cancer risk prediction model in female non-smokers].

Objective: To develop a lung cancer risk prediction model for female non-smokers. Methods: Based on the Kailuan prospective dynamic cohort (2006.05-2015.12), a nested case-control study was conducted. Participants diagnosed with primary pathologically confirmed lung cancer during follow-up were identified as the case group, and others were identified as the control group. A total of 24 701 subjects were included in the study, including 86 lung cancer cases and 24 615 control population, respectively. Questionnaires, physical examinations, and laboratory tests were conducted to collect relevant information. Multivariable-adjusted logistic regressions were conducted to develop a lung cancer risk prediction model. Area Under the Curve (AUC) and Hosmer-Lemeshow tests were used to evaluate discrimination and calibration, respectively. Ten-fold cross-validation was used for internal validation. Results: Two sets of models were developed: the simple model (including age and monthly income) and the metabolic index model [including age, monthly income, fasting blood glucose (FBG), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C)].The AUC (95%CI) [0.745 (0.719-0.771)] of the metabolic index model was higher than that of the simple prediction model [0.688 (0.660-0.716)] (P=0.004). Both the simple model (PHL=0.287) and the metabolic index model (PHL=0.134) were well-calibrated. The results of ten-fold cross-validation indicated sufficient stability, with an average AUC of 0.699 and a standard error (SD) of 0.010. Conclusion: By incorporating metabolic markers, accurate and reliable lung cancer risk prediction model for female non smokers could be developed.

[The development and validation of risk prediction model for lung cancer: a systematic review].

Objective: To systematically understand the global research progress in the construction and validation of lung cancer risk prediction models. Methods: "lung neoplasms" , "lung cancer" , "lung carcinoma" , "lung tumor" , "risk" , "malignancy" , "carcinogenesis" , "prediction" , "assessment" , "model" , "tool" , "score" , "paradigm" , and "algorithm" were used as search keywords. Original articles were systematically searched from Chinese databases (CNKI, and Wanfang) and English databases (PubMed, Embase, Cochrane, and Web of Science) published prior to December 2018. The language of studies was restricted to Chinese and English. The inclusion criteria were human oriented studies with complete information for model development, validation and evaluation. The exclusion criteria were informal publications such as conference abstracts, Chinese dissertation papers, and research materials such as reviews, letters, and news reports. A total of 33 papers involving 27 models were included. The population characteristics of all included studies, study design, predicting factors and the performance of models were analyzed and compared. Results: Among 27 models, the number of American-based, European-based and Asian-based model studies was 12, 6 and 9, respectively. In addition, there were 6 Chinese-based model studies. According to the factors fitted into the models, these studies could be divided into traditional epidemiological models (11 studies), clinical index models (6 studies), and genetic index models (10 studies). 15 models were not validated after construction or were cross-validated only in the internal population, and the extrapolation effect of models was not effectively evaluated; 8 models were validated in single external population; only 4 models were verified in multiple external populations (3-7); the area under the curve (AUC) of models ranged from 0.57 to 0.90. Conclusion: Research on risk prediction models for lung cancer is in development stage. In addition to the lack of external validation of existing models, the exploration of potential clinical indicators was also limited.

IMP3 promotes TNBC stem cell property through miRNA-34a regulation.

OBJECTIVE: To explore the expression and function of insulin-like growth factor II (IGFII) mRNA binding protein (IMP3) in the Triple Negative Breast Cancer (TNBC). MATERIALS AND METHODS: According to previously reported gene expression array, we found that IMP3 had significantly higher expression in the CD44+CD24-ESA+ cell cluster, tumor initiating cell or cancer stem cell (CSCs), compared to other tumor cells. Based on the GEO database (GEO accession No. GSE6883), we detected the mRNA levels of IMP 1,2 and 3 by quantitative polymerase chain reaction (q-PCR) in CD44+CD24-ESA+ cell cluster and other breast tumor cell clusters. Besides, we measured IMP3 expression in microsphere of breast cancer, which exerted more significant tumor stem cell properties. The effects of IMP3 on breast cancer cell stem cell properties were studied by RNA interference and overexpression approaches in vitro. Furthermore, we predicted and identified microRNA, which could target and regulate IMP3 from bioinformatics analysis, and verified the interaction by luciferase assays and rescue experiments. RESULTS: Previously reported data showed that IMP3 expression was significantly upregulated in CD44+CD24-ESA+ cell cluster from breast cancer tissues. Besides, we found IMP3 had higher expression in mesenchymal cells rather than epithelial cells, which was also significantly elevated in SUM159 and T49D cell lines cultured as microsphere rather than adherent cells or differentiated cells. CD44+CD24-ESA+ cell cluster proportion was significantly decreased after silencing IMP3 in SUM1315, and its ability to develop into microsphere was significantly inhibited. By re-expressing IMP3 in SUM315, we restored the self-renewal capacity and tumorigenesis potential of SUM315. Through relative predicting website, we found several miRNAs which could regulate IMP3. miR-34a with highest score was chosen for further analysis. Mimicking miR-34a significantly downregulated IMP3 expression and inhibited its ability to develop into microsphere, while overexpressing IMP3 could rescue this process. CONCLUSIONS: IMP3 plays a vital role in maintaining stem cell properties of breast cancer cells, which could be regulated by mir-34a.

[Lack of progesterone receptor expression predicts poor prognosis in patients with operable ER-positive invasive breast cancer].

OBJECTIVE: To investigate the impact of lack of progesterone receptor (PR) expression on the prognosis of patients with operable ER (estrogen receptor)-positive invasive breast cancer. METHODS: We retrospectively analyzed the clinicopathological features, treatment and survival data of 318 women with ER+ /PR+ and ER+ /PR- invasive breast cancer. RESULTS: Among the 318 patients, there were 219 PR-positive and 99 PR-negative cases. The 5-year overall survival (OS) rate was 92.5%, and the 5-year disease-free survival (DFS) rate was 87.2% in the 318 ER-positive patients. Among them, the 5-year OS rates were significantly different between the PR-positive group (94.6%) and PR-negative group (87.8%, P=0.020), and the 5-year DFS rates were also significantly different from each other (89.8% and 81.6%, respectively, P=0.019). Univariate analysis showed that PR status, tumor size, T stage, axillary lymph node metastasis, and clinical stage were prognostic factors for OS (P<0.05 for all). Multivariate analysis showed that lack of PR expression, T stage ≥2, and positive axillary lymph node metastasis were independent risk factors for poor DFS and OS in ER-positive breast cancer patients (P<0.05 for all). Subgroup analysis showed that lack of PR expression was not significant in predicting poor DFS or OS when patients were in stage Ⅰ or with a small tumor (≤2 cm) (P>0.05 for all), and also showed that premenopausal women with PR-negative disease had poorer DFS and OS than PR-positive patients (P<0.05 for both). CONCLUSIONS: Lack of PR expression is an independent risk factor for poor prognosis in patients with operable ER-positive invasive breast cancer, especially in patients with a large tumor (>2 cm), advanced clinical stage (Stage Ⅱ or Ⅲ) or in premenopausal status.

[Distribution of human papillomavirus (HPV) among HPV positive cervical adenocarcinoma cases detected by laser capture microdissection(LCM)].

OBJECTIVE: To investigate the distribution of human papillomavirus (HPV) in the diseased areas cut from HPV-positive cervical adenocarcinoma (ADC) detected by laser capture microdissection (LCM). METHODS: Paraffin-embedded specimens diagnosed as ADC between 2005 and 2010 were collected from 9 hospitals in 7 regions across China. HPV genotyping was conducted on paraffin sections using sandwich technique and LCM in order to identify HPV infection in the tumor tissues. HE and p16 immunohistochemistry staining were performed to make histological diagnosis. RESULTS: A total of 169 cervical adenocarcinoma cases were recruited, including 94 cases of mucinous adenocarcinoma (ADC-CX), 9 cases of adenosquamous carcinoma (ASC), 19 cases of minimal deviation adenocarcinoma (ADC-MIN), 14 cases of clear cell adenocarcinoma (ADC-CC), 8 cases of endometrioid adenocarcinoma (ADC-ENDO), 9 cases of serous adenocarcinoma (ADC-SER) and 16 cases of adenocarcinoma not otherwise specified (ADC-NOS). Fourteen types of high risk HPV were detected in the whole tissue section (WTS). HPV16 was the most common type, and the second was HPV18 and HPV52, respectively. Compared with WTS, the HPV-positive rate detected by LCM was lower. The HPV positive rates were significantly different among different subtypes of cervical adenocarcinoma (P<0.001). After LCM, the HPV positive rate was 50.8% and 66.7% in the single infection and multiple, infection groups respectively (P=0.14). The positive rates of p16 was significantly different among different subtypes of cervical adenocarcinoma (P<0.001). p16-positive rate was 73.9% in the HPV-positive samples after LCM, significantly higher than the 38.5% of negative samples (P<0.001). CONCLUSIONS: Laser capture dissection technique can more precisely reflect the HPV distribution in cervical adenocarcinomas. The etiological association between HPV infection and cervical adenocarcinoma occurrence is not as close as that reported in the literature.

Alteration of bone cell function by RANKL and OPG in different in vitro models.

BACKGROUND: Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) are well-documented potent regulators of osteoclast development. However, their effects in mature bone cells and in organ cultures have not been well studied. It is uncertain whether their activities in different experimental models are comparable. MATERIALS AND METHODS: RANKL and OPG were evaluated for their activities in mouse calvarial organ cultures, mouse bone marrow cultures, isolated rat mature osteoclast assays and rat primary osteoblast cultures. Results In murine calvarial organ culture, both muRANKL (> or = 10 ng mL(-1)) and rRANKL (> or = 100 ng mL(-1)) significantly stimulated (45)Ca release, while OPG (> or = 50 ng mL(-1)) was an inhibitor of bone resorption. Meanwhile, [(3)H]-thymidine incorporation in this assay was also modulated (indicating proliferation increases in the osteoblast lineage of cells) although these peptides had no direct effect on [(3)H]-thymidine incorporation in isolated osteoblast assays. In mouse bone marrow cultures, muRANKL (> or = 1 ng mL(-1)) and rRANKL (> or = 5 ng mL(-1)) significantly stimulated osteoclastogenesis. The number of nuclei per osteoclast was also significantly increased. OPG strongly inhibited this index, with over 90% suppression at 1 ng mL(-1). Both muRANKL (10 ng mL(-1)) and rRANKL (100 ng mL(-1)) stimulated, while OPG (10 ng mL(-1)) inhibited osteoclast activity in isolated mature osteoclast assays. CONCLUSION: The current study demonstrated that bone resorption modulated by RANKL and OPG, in murine calvarial organ culture, leads to changes in osteoblast proliferation, suggesting a feedback mechanism from osteoclasts to osteoblasts. In addition, it was found that RANKL and OPG have more potent effects on osteoclastogenesis than on the activity of mature osteoclasts.

Id-1, ITF-2, and Id-2 comprise a network of helix-loop-helix proteins that regulate mammary epithelial cell proliferation, differentiation, and apoptosis.

Mammary epithelial cells proliferate, invade the stroma, differentiate, and die in adult mammals by mechanisms that are poorly understood. We found that Id-1, an inhibitor of basic helix-loop-helix transcription factors, regulates mammary epithelial cell growth, differentiation, and invasion in culture. Here, we show that Id-1 is expressed highly during mammary development in virgin mice and during early pregnancy, when proliferation and invasion are high. During mid-pregnancy, Id-1 expression declined to undetectable levels as the epithelium differentiated fully. Surprisingly, Id-1 increased during involution, when the epithelium undergoes extensive apoptosis. To determine whether Id-1 regulates both proliferation and apoptosis, we constitutively expressed Id-1 in mammary epithelial cell cultures. Id-1 stimulated proliferation in sparse cultures but induced apoptosis in dense cultures, which reflect epithelial cell density during early pregnancy and involution, respectively. To understand how Id-1 acts, we screened a yeast two-hybrid library from differentiating mammary epithelial cells and identified ITF-2, a basic helix-loop-helix transcription factor, as an Id-1-interacting protein. Overexpression of ITF-2 significantly reduced Id-1-stimulated proliferation and apoptosis. We show further that, in contrast to Id-1, Id-2 was expressed highly in differentiated mammary epithelial cells in vivo and in culture. In culture, Id-2 antisense transcripts blocked differentiation. Our results suggest that Id-1, ITF-2, and Id-2 comprise a network of interacting molecular switches that govern mammary epithelial cell phenotypes.

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus.

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

A role for Id-1 in the aggressive phenotype and steroid hormone response of human breast cancer cells.

The helix-loop-helix protein Id-1 inhibits the activity of basic helix-loop-helix transcription factors, and is an important regulator of cell growth and tissue-specific differentiation. We have shown (P. Y. Desprez et al., Mol. Cell. Biol., 18: 4577-4588, 1998) that ectopic expression of Id-1 inhibits differentiation and stimulates the proliferation and invasiveness of mouse mammary epithelial cells, and that there is a correlation between the levels of Id-1 protein and the aggressiveness of several human breast cancer cell lines. Here, we show that aggressive and metastatic breast cancer cells express high levels of Id-1 mRNA because of a loss of serum-dependent regulation that is mediated by a 2.2-kb region of the human Id-1 promoter. Three lines of evidence suggest that unregulated Id-1 expression may be an important regulator of the aggressive phenotype of a subset of human breast cancer cells: (a) a constitutively expressed Id-1 cDNA, when introduced into a nonaggressive breast cancer cell line (T47D), conferred a more aggressive phenotype, as measured by growth and invasiveness; (b) Id-1 was an important mediator of the effects of sex steroid hormones on T47D cell proliferation. Estrogen stimulated proliferation and induced Id-1 expression, whereas progesterone inhibited proliferation and repressed Id-1 expression. Progesterone repressed Id-1 expression, at least in part by repressing transcription. Most importantly, an antisense oligonucleotide that reduced Id-1 protein levels reduced the ability of estrogen to stimulate cell proliferation, whereas constitutive Id-1 expression rendered cells refractory to growth inhibition by progesterone; and (c) using a limited number of breast cancer biopsies, we showed that Id-1 was more frequently expressed in infiltrating carcinomas compared with ductal carcinomas in situ. Our results suggest that Id-1 can control the malignant progression of breast cancer cells, particularly that mediated by sex steroid hormones. Moreover, Id-1 has the potential to serve as a marker for aggressive breast tumors.

Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes.

Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.

Comparison of the effects of calcitonin gene-related peptide and amylin on osteoblasts.

Calcitonin gene-related peptide (CGRP) and amylin are homologous 37-amino-acid peptides which have been demonstrated to have anabolic effects on bone. It is not clear whether these effects are mediated by a common receptor, nor is it known which ligand is the more potent. These questions are addressed in the present study using cultures of fetal rat osteoblasts. CGRP increased cell number when present in a concentration >/=10-9 M, but 10-8 M CGRP was required to stimulate thymidine and phenylalanine incorporation. Amylin was effective on these indices at 100-fold lower concentrations, and its maximal effects were about twice as great as those of CGRP. ED50's for the effects of amylin and CGRP on cell number were 10-12 M and 10-10 M, respectively. There was no additivity between maximal doses of the peptides on these indices. The effects of specific receptor blockers on the maximal stimulation of cell number by these peptides were also studied. The CGRP receptor-blocker, CGRP-(8-37), completely blocked the effect of CGRP at blocker concentrations >/=10-9 M. In contrast, the amylin receptor blocker, amylin-(8-37), completely blocked the effects of CGRP when the blocker was present in concentrations as low as 10-11 M. The KI of CGRP-(8-37) was 2 x 10-10 M and that of amylin-(8-37) was 7 x 10-12 M. In converse experiments studying the blockade of maximal doses of amylin, amylin-(8-37) 10-10 M was effective (KI 1 x 10-10 M), whereas a 100-fold greater concentration of CGRP-(8-37) was necessary to achieve the same effect (KI 6 x 10-9 M). It is concluded that amylin and CGRP probably act through a common receptor to stimulate osteoblast growth, and that this receptor has a higher affinity for amylin than for CGRP.

Regulation of mammary epithelial cell phenotypes by the helix-loop-helix protein, Id-1.

Mammary epithelial cells undergo cycles of proliferation, invasion, differentiation and apoptotic cell-death throughout adult life. The molecular mechanisms that regulate these complex and co-ordinated developmental programs are poorly understood. We have identified Id-1 protein, a negative regulator of basic helix-loop-helix transcription factors, as a critical regulator of these normal mammary epithelial cell phenotypes. We also found that Id-1 is an important regulator of the aggressive and invasive phenotype, as well as mediator of the effects of sex steroid hormones, in human breast cancer cells.

A novel pathway for mammary epithelial cell invasion induced by the helix-loop-helix protein Id-1.

Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a approximately 120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of approximately 120 kDa was expressed by the mammary gland during involution, a time when Id-1 expression is high and there is extensive tissue remodeling. Moreover, high levels of Id-1 expression and the activity of a approximately 120-kDa gelatinase correlated with a less-differentiated and more-aggressive phenotype in human breast cancer cells. We suggest that Id-1 controls invasion by normal and neoplastic mammary epithelial cells, primarily through induction of a approximately 120-kDa gelatinase. This Id-1-regulated invasive phenotype could contribute to involution of the mammary gland and possibly to the development of invasive breast cancer.

Dissociation of the effects of amylin on osteoblast proliferation and bone resorption.

This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 x 10(-11) and 2.4 x 10(-10) M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.

A unique intronic splicing enhancer controls the inclusion of the agrin Y exon.

Alternative splicing of the agrin mRNA controls the ability of agrin protein to induce the clustering of acetylcholine receptors at the neuromuscular junction. Using a transfectable reporter gene, we show that one agrin alternative exon, the Y exon, is controlled by a regulatory sequence in the downstream intron. Portions of this intronic sequence have the properties of a splicing enhancer that can activate splicing of a heterologous exon when placed in the intron downstream. The regulatory region is complex in structure, containing several different elements capable of activating splicing. Individual enhancing elements differ in their cell-type specificity, and are not apparently synergistic, as two elements together induce lower splicing than either does separately. Essential nucleotides within these regulatory elements were identified by scanning mutagenesis across the active region. Interestingly, the elements do not appear similar to known intronic splicing enhancer elements. This Y exon enhancer and its components take part in an apparent combinatorial system of control where multiple regulatory elements of varying activity combine to produce a precisely cell-specific exon inclusion. As a major contributor to the regulation of the Y exon, the enhancer ultimately controls the properties of the agrin protein.

Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice.

Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-alpha production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with beta-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-alpha is added or overexpressed. Thus, (c) the enhanced TGF-alpha production when cells are not in three-dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-alpha. The data suggest that ECM upregulates WAP by downregulating TGF-alpha production. We also propose that changes in TGF-alpha activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.

Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock.

Tumor necrosis factor-alpha (TNF) is a pluripotent cytokine that mediates many of the hemodynamic manifestations of endotoxic shock. To determine whether TNF is responsible for postburn myocardial dysfunction, we compared cardiac function (Langendorff preparation) in 49 guinea pigs 18 h after thermal injury. Group 1 (n = 15) was sham burned; all remaining animals received a 43% surface area burn under anesthesia. Group 2 (n = 15) received lactated Ringer solution (LR, 4 ml.kg-1.%burn-1). Group 3 (n = 9) received LR and drug vehicle. Group 4 (n = 10) received LR plus 1 mg of TNF inhibitor consisting of the human p80 TNF receptor linked to the Fc portion of human immunoglobulin G1, which was shown to specifically bind and neutralize TNF secreted by guinea pig peritoneal macrophages in vitro. Burn injury caused a significant fall in left ventricular pressure (LVP, from 86 +/- 2 to 62 +/- 3 mmHg, P < 0.05) and maximal rate of LVP rise (+) and fall (-) (+/- dP/dtmax) [from 1,365 +/- 42 to 1,109 +/- 44 mmHg/s (P < 0.05) and from 1,184 +/- 31 to 881 +/- 40 mmHg/s (P < 0.05), respectively], a decrease in time to peak systolic LVP (from 111 +/- 2 to 102 +/- 2 ms, P < 0.05), and a decrease in time to +dP/dtmax (from 57 +/- 1 to 48 +/- 1 ms, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)