Igor B. Buchwalow

Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ

Current classification systems in proliferative mammary gland pathology are based on a two‐cell system, recognizing only glandular and myoepithelial lines of differentiation. A third cell type has recently been characterized in normal breast tissue by double‐immunofluorescence analysis to express cytokeratin 5 (Ck5) only. These cells were shown to represent progenitor or adult stem cells that give rise to the glandular and myoepithelial cell lineage. The double‐labelling technique has been applied to characterize a spectrum of intraductal epithelial proliferations, namely benign usual ductal hyperplasia, atypical ductal hyperplasia, and ductal carcinoma in situ, all of which are thought to represent the gradual steps of a sequence in the development of breast cancer. Immunofluorescence studies with specific antibodies against Ck5, Ck8/18/19, and smooth muscle actin were complemented by western blotting analysis of Ck5 and Ck8/18/19 expression in normal breast tissue and in proliferative lesions. Usual ductal hyperplasia appears to be a Ck5‐positive committed stem (progenitor) cell lesion with the same differentiation potential as seen in the normal breast. This is in sharp contrast to atypical ductal hyperplasia/ductal carcinoma in situ, which display the differentiated glandular immunophenotype (Ck8/18/19‐positive, but Ck5‐negative). These data require the abandonment of the idea of an obligate biological continuum of intraductal proliferations from benign to malignant. This study provides evidence that cells undergoing malignant transformation tend to be fairly advanced in the glandular lineage of differentiation. The committed stem (progenitor) cell model may contribute to a better understanding of both benign proliferative breast disease and breast cancer development. Copyright

Vascular smooth muscle and nitric oxide synthase

The concept of endothelium‐derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP‐dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium‐independent manner.—Buchwalow, I. B., Podzuweit, T., Böcker, W., Samoilova, V. E., Thomas, S., Wellner, M., Baba, H. A., Robenek, H., Schnekenburger, J., Lerch, M. M. Vascular smooth muscle and nitric oxide synthase. FASEB J. 16, 500–508 (2002)

Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin.

The CD4+ natural killer (NK)T cells in the liver are potent IL‐4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL‐4‐producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70 % Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL‐4 to IFN‐γ. The number of CD4+ NK1+ TCRα / βintermediate cells increased in the low‐density fraction (< 40 % Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL‐4‐producing cells, however, was small and high frequencies of IFN‐γ‐secreting cells were identified in the low‐density fraction after TCR/CD3 ligation. Accordingly, selected low‐density CD4+ NKT cells encompassed high numbers of IFN‐γ producers and minute numbers of IL‐4‐secreting cells. Induction of low‐density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL‐12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL‐4 to IFN‐γ through IL‐12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN‐γ‐producing Th1 cells.

Gains and losses of adhesion molecules (CD44, E‐cadherin, and β‐catenin) during oral carcinogenesis and tumour progression

The aim of this study was to define whether or not the impaired expression of CD44, E‐cadherin (E‐cad), and β‐catenin (β‐cat) correlates with the clinical evolution and prognosis of oral cancer. Ninety‐three primary oral squamous cell carcinomas (OSCCs) with tumour‐adjacent normal and/or dysplastic mucosa, 30 associated metastases, and 12 recurrences were immunostained for CD44s, ‐v3, ‐v4, ‐v5, ‐v6, ‐v7, ‐v9, E‐cad, and β‐cat. In non‐neoplastic epithelium, all molecules investigated were constitutively expressed in the basal layers. In the majority of dysplasias, immunoreactivity for all adhesion molecules was increased, but there was restricted loss for CD44s, E‐cad, and β‐cat in a few cases. In carcinomas, a striking accumulation of CD44s, v3, v4, v9 and a loss of E‐cad/β‐cat were observed at the invasive tumour front. In metastases and recurrences, besides a loss of CD44s, v4, v7, and E‐cad, a significant increase of v9 was recorded, whereas CD44v5 and v6 remained unchanged. Clinically, reduced expression of CD44v3, E‐cad, and changes of CD44v9 phenotype within the primary tumours correlated significantly with poor prognosis; decreased β‐cat expression was a predictive marker for nodal metastases. These findings indicate that there is some perturbed expression of adhesion molecules during the stepwise course of oral carcinogenesis and tumour progression. Distinct phenotypic alterations project poor prognosis, while others predict metastasis. Some of these restricted molecular changes may serve as potential targets for future antibody‐based tumour therapy. Copyright

Inducible nitric oxide synthase in the myocard.

Recognition of significance of nitric oxide synthases (NOS) in cardiovascular regulations has led to intensive research and development of therapies focused on NOS as potential therapeutic targets. However, the NOS isoform profile of cardiac tissue and subcellular localization of NOS isoforms remain a matter of debate. The aim of this study was to investigate the localization of an inducible NOS isoform (NOS2) in cardiomyocytes. Employing a novel immunocytochemical technique of a catalyzed reporter deposition system with tyramide and electron microscopical immunocytochemistry complemented with Western blotting and RT-PCR, we detected NOS2 both in rat neonatal and adult cultured cardiomyocytes and in the normal myocard of adult rats as well as in the human myocard of patients with dilative cardiomyopathy. NOS2 was targeted predominantly to a particulate component of the cardiomyocyte – along contractile fibers, in the plasma membrane including T-tubules, as well as in the nuclear envelope, mitochondria and Golgi complex. Our results point to an involvement of NOS2 in maintaining cardiac homeostasis and contradict to the notion that NOS2 is expressed in cardiac tissue only in response to various physiological and pathogenic factors. NOS2 targeting to mitochondria and contractile fibers suggests a relationship of NO with contractile function and energy production in the cardiac muscle.

Non-specific binding of antibodies in immunohistochemistry: fallacies and facts

The current protocols for blocking background staining in immunohistochemistry are based on conflicting reports. Background staining is thought to occur as a result of either non-specific antibody (Ab) binding to endogenous Fc receptors (FcRs) or a combination of ionic and hydrophobic interactions. In this study, cell and tissue samples were processed according to routine protocols either with or without a blocking step (goat serum or BSA). Surprisingly, no Abs in samples processed without a blocking step showed any propensity for non-specific binding leading to background staining, implying that endogenous FcRs do not retain their ability to bind the Fc portion of Abs after standard fixation. Likewise, we did not find any non-specific Ab binding ascribable to either ionic or hydrophobic interactions. We determined that traditionally used protein blocking steps are unnecessary in the immunostaining of routinely fixed cell and tissue samples.

Impaired relaxation in transgenic mice overexpressing junctin

OBJECTIVE Junctin is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum, which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), triadin, and calsequestrin. METHODS To better understand the role of junctin in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of junctin to mouse heart, using the alpha-MHC promoter to drive protein expression. RESULTS The protein was overexpressed 10-fold in mouse ventricles and overexpression was accompanied by cardiac hypertrophy (19%). The levels of two other junctional SR-proteins, the ryanodine receptor and triadin, were reduced by 32% and 23%, respectively. However, [3H]ryanodine binding and the expression levels of calsequestrin, phospholamban and SERCA2a remained unchanged. Cardiomyocytes from junctin-overexpressing mice exhibited impaired relaxation: Ca(2+) transients decayed at a slower rate and cell relengthening was prolonged. Isolated electrically stimulated papillary muscles from junctin-overexpressing hearts exhibited prolonged mechanical relaxation, and echocardiographic parameters of relaxation were prolonged in the living transgenic mice. The amplitude of caffeine-induced Ca(2+) transients was lower in cardiomyocytes from junctin-overexpressing mice. The inactivation kinetics of L-type Ca(2+) channel were prolonged in junctin-overexpressing cardiomyocytes using Ca(2+) or Ba(2+) as charge carriers. CONCLUSION Our data provide evidence that cardiac-specific overexpression of junctin is accompanied by impaired myocardial relaxation with prolonged Ca(2+) transient kinetics on the cardiomyocyte level.

Different proliferative activity of the glandular and myoepithelial lineages in benign proliferative and early malignant breast diseases

The aim of the present study was to explore cell biological characteristics of normal breast, benign proliferative breast diseases and noninvasive breast malignancies based on the recently published adult progenitor cell concept from our group. Here, we investigated the proliferative activity of CK5/14+, CK8/18/19+ and α-smooth muscle actin+ cellular phenotypes encountered in normal mammary gland, in a series of usual ductal hyperplasias and early malignant breast diseases, such as atypical ductal and lobular hyperplasias, as well as ductal and lobular in situ carcinomas. Immunohistochemical double labeling was performed on frozen sections from diagnostic breast biopsies by using antibodies to basal cytokeratins (CK5/14), glandular cytokeratins (CK8/18/19), smooth muscle actin and the Ki-67 antigen (MIB1). Normal breast tissues and usual ductal hyperplasias were characterized by a heterogeneous cellular composition of the growth fraction. The proliferative cell compartment consisted of CK8/18/19+ glandular and, in a variable proportion, CK5/14+ progenitor phenotypes. In contrast, noninvasive breast malignancies were composed of a monotonous proliferation of CK 8/18/19+ neoplastic glandular cells. These findings indicate a significant role of progenitor cells in the development of benign proliferative breast diseases and lend support to the view that malignant transformation in the human breast usually occurs in a cell committed to the glandular lineage. Our results provide cell kinetic support to the functional progenitor cell hypothesis, and we propose this concept as an operative model for understanding benign proliferative and malignant breast diseases.

Critical Role of Transcription Factor Cyclic AMP Response Element Modulator in β1-Adrenoceptor–Mediated Cardiac Dysfunction

Background— Chronic stimulation of the &bgr;1-adrenoceptor (&bgr;1AR) plays a crucial role in the pathogenesis of heart failure; however, underlying mechanisms remain to be elucidated. The regulation by transcription factors cAMP response element-binding protein (CREB) and cyclic AMP response element modulator (CREM) represents a fundamental mechanism of cyclic AMP–dependent gene control possibly implicated in &bgr;1AR-mediated cardiac deterioration. Methods and Results— We studied the role of CREM in &bgr;1AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of &bgr;1AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in &bgr;1AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1&agr;, and cardiac &agr;-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient &bgr;1AR-transgenic hearts. Conclusions— The results imply the regulation of genes by CREM as an important mechanism of &bgr;1AR-induced cardiac damage in mice.

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes.

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.