High copy number variation of cancer-related microRNA genes and frequent amplification of DICER1 and DROSHA in lung cancer

A growing body of evidence indicates that miRNAs may be a class of genetic elements that can either drive or suppress oncogenesis. In this study we analyzed the somatic copy number variation of 14 miRNA genes frequently found to be either over- or underexpressed in lung cancer, as well as two miRNA biogenesis genes, DICER1 and DROSHA, in non-small-cell lung cancer (NSCLC). Our analysis showed that most analyzed miRNA genes undergo substantial copy number alteration in lung cancer. The most frequently amplified miRNA genes include the following: miR-30d, miR-21, miR-17 and miR-155. We also showed that both DICER1 and DROSHA are frequently amplified in NSCLC. The copy number variation of DICER1 and DROSHA correlates well with their expression and survival of NSCLC and other cancer patients. The increased expression of DROSHA and DICER1 decreases and increases the survival, respectively. In conclusion, our results show that copy number variation may be an important mechanism of upregulation/downregulation of miRNAs in cancer and suggest an oncogenic role for DROSHA.     

The VEGFR2, COX‐2 and MMP‐2 polymorphisms are associated with clinical outcome of patients with inoperable non‐small cell lung cancer

Certain common inherited variations in genes involved in tumor angiogenesis, progression and metastasis may contribute to cancer therapy outcome and prognosis by altering the gene expression and protein activity. In this report, we examined the effect of functional polymorphisms in MMP‐1, MMP‐2, MMP‐3, VEGF, VEGFR2, FGFR4 and COX‐2 genes on overall (OS) and progression‐free survival (PFS) of 350 Caucasian patients with inoperable non‐small cell lung cancer (NSCLC). The results of multivariate analysis indicated that VEGFR2 ‐906C and COX‐2 ‐1195G alleles were strongly associated with poor OS and PFS (p = 0.002 and 0.015, respectively, for OS; p = 0.009 and 0.015, respectively, for PFS), while MMP‐2 ‐1306 T allele carriers had significantly reduced PFS (p = 0.010). Moreover, an increased risk of death and progression was significantly associated with the number of adverse alleles for VEGFR2/COX‐2 (p = 0.0005 for OS and 0.0006 for PFS in >1 adverse allele carriers) and VEGFR2/COX‐2/MMP‐2 combinations (p = 0.0003 for OS and 0.0001 for PFS in patients with >2 adverse alleles). Finally, VEGFR2 TC/CC, COX‐2 AG/GG and MMP‐2 CT/TT genotypes as well as “at risk” allele combinations were identified as independent predictors of unfavorable OS and PFS in the group. In conclusion, the data suggest that selected VEGFR2, COX‐2 and MMP‐2 polymorphisms may be potential prognostic markers in unresectable NSCLC treated with radiotherapy with or without chemotherapy, although further validation studies are warranted to confirm our observations.     

Impaired coronary flow and left ventricular dysfunction after mechanical recanalization in acute myocardial infarction: role of neurohumoral activation?

Background: Reopening of the infarct-related coronary artery is the treatment of choice in the clinical setting of acute myocardial infarction. Nevertheless the removal of the total occlusion obtained either by thrombolysis or by primary angioplasty is followed by the ischemia/reperfusion sequelae. One of many proposed mechanisms playing a role in ischemia/reperfusion damage is a persistent increase in vasoconstrictor tone, which reduces cardiac function and impairs myocardial blood flow during primary percutaneous coronary intervention in acute myocardial infarction (PAMI). Methods: To investigate early neurohumoral changes during PAMI we enrolled 18 patients, who were collated to 13 patients with stable angina undergoing elective PTCA. To evaluate angiotensin II (AngII), endothelin-1 (ET-1), vasopressin (AVP), norepinephrine (NE), troponin T (TNT), creatinephosphate kinase (CPKM) and isoenzyme MB (CPKMB), we collected blood from the pulmonary artery before and immediately after the infarct-related artery (IRA; TIMI 0 → 2–3) or culprit lesion revascularization. Hemodynamic and angiographic LV-function parameters were compared to biochemical data. Corrected TIMI-frame count (CTFC) was used as an index of coronary blood flow and correlated to the biochemical measurements. Results: CTFC in the IRA correlated inversely (p = 0.03; r = −0.51) with left ventricular ejection fraction measured after 10 days, and positively (p = 0.03; r = 0.54) with the maximal amount of LDH released after onset of AMI. There was an abrupt and long lasting rise in ET-1 (+65 %; p < 0.001) and an instant short lasting increase in AVP (+37 %; p < 0.05), whereas NE concentrations were elevated prior to PAMI and remained elevated during reperfusion. Correlations with CTFC were found for ET-1 (p = 0.01; r = 0.61) and NE (p = 0.01; r = 0.58) during reperfusion. The extent of left ventricular dysfunction correlated with the concentrations of AVP and NE during reperfusion. Conclusions: There is evidence for a distinct pattern of neurohumoral activation during early reperfusion in acute myocardial infarction. In particular, we documented substantial increases in AVP and ET-1. Left ventricular wall-stress appears to be involved in the release of AVP. Elevated levels of ET-1 and NE are associated with impaired angiographic reperfusion and increased myocardial damage after mechanical recanalization. Received: 13 December 2001, Returned for 1. revision: 14 January 2002, 1. Revision received: 10 April 2002, Returned for 2. revision: 30 April 2002, 2. Revision received: 10 May 2002, Accepted: 13 May 2002     

Myocardial catecholamine responsiveness of spontaneously hypertensive rats as influenced by swimming training

Summary Alterations of myocardial mechanical catecholamine responsiveness by swimming training (2×90 min/day, 4 weeks) were examined in 13-week-old spontaneously hypertensive male rats (SHR). The relationships between myocardial mechanical catecholamine responsiveness and ventricular -adrenoceptors as well as myosin isoenzyme pattern were also examined. Compared with sedentary controls, trained rats showed a greater responsiveness to isoproterenol (10^–6 mol/l) on isometric tension (T) and its first derivative (dT/dt) (T:0.45±0.55 vs. –0.15±0.11 10^–2 N/mm², p<0.01, dT/dt: 17.1±10.1 vs. 8.3±3.6 10^–2 N/mm²·s, p<0.05). In sedentary SHR, dT/dt_max increased significantly, whereas developed tension decreased slightly, coupled with a decrease of time to peak tension by high dose (10^–6 mol/l) isoproterenol. Therefore, it can be stated that dT/dt is a better indicator for catecholamine sensitivity than isometric tension. -adrenoceptor density ([³H]-dihydroalprenolol binding) decreased significantly in trained rats (68.7±7.62 vs. 102.4±4.37 fmol/mg protein, p<0.01) with no significant difference in K_D values (4.61±2.26 vs. 6.11±1.94 nM, ns). In addition, myosin isoenzyme pattern revealed by pyrophosphate gel electrophoresis shifted towards VM-1 after swimming training. The increased catecholamine sensitivity of fast contracting myocardium is, in principle, compatible with the assumption of cAMP-dependent regulation of myofibrillar ATPase activity (21) or cross bridge kinetics (9), although other postreceptor processes should also be taken into consideration for the increased catecholamine sensitivity.Supported by the Deutsche Forschungsgemeinschaft     

Differential expression of AT1 receptors in the pituitary and adrenal gland of SHR and WKY

The renin-angiotensin (ANG) system has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR). Because SHR are more susceptible to stress than normotensive Wistar-Kyoto rats (WKY), we measured the mRNA expression of AT1A, AT1B, and AT2 receptors in the hypothalamo-pituitary-adrenal (stress) axis of male SHR in comparison to age-matched WKY at prehypertensive (3 to 4 weeks), developing (7 to 8 weeks), and established (12 to 13 weeks) stages of hypertension. AT1A receptor mRNA was mainly expressed in the hypothalamus and adrenal gland. AT1B receptor mRNA was detected in the pituitary and adrenal gland. AT2 receptor mRNA was prominent only in the adrenal gland. When compared with WKY, SHR showed increased AT1A receptor mRNA levels in the pituitary gland at all ages in contrast to reduced pituitary AT1B receptor mRNA levels. In the adrenal gland of SHR, AT1B receptor mRNA levels were decreased at the hypertensive stages when compared with WKY. The reduced expression of adrenal AT1B receptor mRNA was localized selectively in the zona glomerulosa by in situ hybridization. No differences were observed between WKY and SHR in the expression of hypothalamic ANG receptors. ANG significantly increased plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in dexamethasone-treated SHR but not in WKY. The aldosterone response to ANG was similar in SHR and WKY. Our results suggest a differential gene expression of AT1A and AT1B receptors in the hypothalamo-pituitary-adrenal axis of SHR and normotensive WKY and imply the participation of AT1 receptors in an exaggerated endocrine stress response of SHR to ANG.     

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.It is well accepted that direct virus neutralization is an important element of antibody-mediated protection against HIV-1 (refs. 1–6 and reviewed in ref. 7). In contrast, less is known about the role of Fc-mediated effector function in the control of HIV-1, although four lines of evidence signal its importance. First, studies in HIV-1–infected people (8–14) and in macaques infected with simian immunodeficiency virus (15, 16) consistently show an inverse correlation between Fc-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) (8, 9) or antibody-dependent cell-mediated viral inhibition (ADCVI), and viral loads or decreased disease progression (17). Second, vaccine-elicited protection both in nonhuman primates (18–21) and in a subset of human subjects in the Vax-004 trial (22) correlates with Fc-mediated effector function often observed in the absence of detectable neutralizing antibodies (18–21). Similarly, there was an inverse relationship between acquisition of HIV-1 and ADCC in the RV144 trial for a subset of subjects who had low to moderate IgA anti-gp120 titers (23). Third, breast milk IgG ADCC responses to gp120 but not to virus neutralization correlated with reduced perinatal transmission of HIV-1 (24). Fourth, passive immunization studies in nonhuman primates (25, 26) showed that abrogation of Fc-mediated effector function diminished the sterilizing protection afforded by the neutralizing mAb b12. These compelling studies show that neutralization alone significantly protects against a simian-human immunodeficiency virus challenge and that Fc-mediated effector function augments this effect. Taken together, these four lines of investigation strongly suggest that Fc-mediated effector function in addition to neutralization contributes to antibody-mediated protection against HIV-1. Thus, it is important to determine the precise relationships among antibody specificity, neutralization, and Fc-mediated effector function in protection against HIV-1.In this report, we probe these relationships using a panel of human mAbs that recognize transitional epitopes exposed during the earliest stage of viral entry, the interaction of gp120 with CD4. Our studies deliberately focus on antibody responses to epitopes that become exposed during viral entry because passive immunization studies indicate that an antibody has at most a 24-h window to block transmission (ref. 27; reviewed in ref. 28). Thus, transmission-blocking antibodies must block infection by direct neutralization of HIV-1, by Fc-mediated killing of nascently infected cells, or both. Although these two effector functions often are coincident for a given mAb specificity (29, 30), they can be dissociated because nonneutralizing epitopes on both gp120 (12, 31) and gp41 (32) can be ADCC targets. In this report, we probe the relationships among antibody specificity, ADCC, and neutralization using a panel of human mAbs that recognize transitional epitopes exposed on target cells during viral entry.