Design, synthesis, computational calculation and biological evaluation of some novel 2-thiazolyl hydrazones.

In the present study a novel series of 1-(1-(4-isobutylphenyl)ethylidene)-2-(4-phenylthiazol-2-yl)hydrazine 2a and its derivatives 2b-2f have been synthesized by the cyclization of 1-(1-(4-isobutylphenyl)ethylidene)thiosemicarbazide with 2-bromoacetophenone/ 4-substituted 2-bromoacetophenones. The structures of the synthesized thiazolyl hydrazones 2a-2f were characterized by FT-IR, (1)H, (13)C NMR, 2D NMR and mass spectral techniques. The molecular geometries were also investigated theoretically using B3LYP functional with 6-311G(d,p) basis set. To explain the molecular properties energy gap (Eg), electronegativity (χ), hardness (g), electrophilicity (ω) and softness (S) were computed, natural bonding orbital (NBO) analysis and molecular electrostatic potential (MEP) were also performed at the same level of theory. All the synthesized thiazolyl hydrazones 2a-2f were screened for their in vitro antimicrobial activity against selected bacterial and fungal strains. The results showed that the heterocyclic thiazolyl hydrazone derivatives exhibit a promising selective inhibitory activity against various bacterial and fungal strains.

1-Diphenyl-methyl-ene-2-(9H-fluoren-9-yl-idene)hydrazine.

In the title mol-ecule, C(26)H(18)N(2), the 9H-fluorene unit is almost planar, as the cyclo-penta-diene ring makes dihedral angles of 1.12 (6) and 1.46 (6)° with the fused benzene rings. The dihedral angle between the two phenyl rings of the diphenyl-methyl-ene residue is 61.78 (6)°.

(E)-1-Diphenyl-methyl-idene-2-[(1H-indol-3-yl)methyl-idene]hydrazine.

In the title compound, C(22)H(17)N(3), the 1H-indole unit is essentially planar, with a dihedral angle of 0.95 (10)° between the pyrrole ring and the fused benzene ring. The dihedral angle between the two phenyl rings is 65.09 (10)°. In the crystal, an inter-molecular N-H⋯N hydrogen bond forms an infinite chain in the b-axis direction.

Endosomal colocalization of melanocortin-3 receptor and beta-arrestins in CAD cells with altered modification of AKT/PKB.

The melanocortin 3-receptor is involved in regulating energy metabolism, body fluid composition and inflammatory responses. Melanocortin receptors function by activating membrane bound adenylate cyclase. However, the literature reports indicate that some G protein coupled receptors (GPCRs) can also activate mitogen activated protein kinase (MAPK) or phosphoinositide 3 kinase (PI3K) signaling pathways consequent to their endocytosis. These studies were undertaken to evaluate the role of these pathways in MC3R signaling in brain-stem neuronal cells. Recruitment of arrestins is implicated in the activation of secondary pathways by GPCRs and our data shows the colocalization of either arrestin B1 or B2 with MC3R in endosomes. An alteration in PKB phosphorylation pattern was observed in MC3R expressing cells independent of agonist stimulation. MC3R transfectants exhibited increased proliferation rates and inhibition of PKB pathway with triciribine abrogated cell proliferation in both vector control and MC3R transfectants. PKB is constitutively active in proliferating CAD cells but could be further activated by culturing the cells in differentiation medium. These studies suggest that the AKT/PKB pathway plays an important role in the proliferation of CAD cells and suggest a link between MC3R and cell growth pathways that may involve the alteration of AKT/PKB signaling pathway.

Differential expression of TCEAL1 in esophageal cancers by custom cDNA microarray analysis.

To find new genes involved in esophageal squamous cell carcinogenesis, we constructed custom cDNA arrays and used the arrays to compare gene expression profiles of 12 matched normal and malignant esophageal samples including seven superficial cancer tissues. The arrays represented nearly 4000 genes, including 1728 that were specifically selected based on pilot studies to find genes that were differentially expressed in esophageal cancers. Expression values for all genes were normalized for each sample and were compared in normal versus tumor tissues. There was a marked decrease in the levels of the transcriptional elongation factor A gene in all 12 of the squamous cell cancer samples compared to matched normal samples. Because the transcription elongation factor A gene has not been previously reported to be involved in cancer development, our results suggest that further investigation of its role in esophageal carcinogenesis is warranted.

Functional genomics, gene arrays, and the future of pathology.

The human genome project has attracted a great deal of attention in recent years among the general public as well as the scientific community. Although it is likely to be a number of years before many of the expected benefits of the genomics revolution are realized, the impact of these scientific breakthroughs on diagnostic pathology is likely to become apparent relatively quickly. In particular, gene array technology, which allows gene expression measurements of thousands of genes in parallel, provides a powerful tool for pathologists seeking new markers for diagnosis. Several recent studies demonstrate how the gene array approach can not only recognize markers for known categories of neoplasia but also lead to recognition of different categories not previously appreciated. Although this approach shows great potential, the successful application of gene arrays to diagnostic problems will require thoughtful interpretation, just as immunochemical technologies require careful planning and analysis.

Spreadsheet-based program for the analysis of DNA methylation.

Methylation of DNA in CpG dense regions of gene promoters (CpG islands) is important for transcriptional inactivation of selective genes in normal and neoplastic cells. Here, we present a spreadsheet-based program adapted from Microsoft Excel that is useful for identifying CpG islands and for assisting in the laboratory analysis of DNA methylation of these regions. Upon execution of the program, a customized workbook analyzes an entered DNA sequence for the total number and percentage cytosine and guanine nucleotides, the total number and percentage of CpG sites, and a CpG:GpC ratio. The program also displays the distribution of CpG sites in a visual format as well as in two different graphical formats. Finally, the program assists in laboratory studies of DNA methylation that employ bisulfite modification of DNA by displaying methylation-dependent effects of bisulfite treatment on DNA sequences.

Classification of small cell lung cancer and pulmonary carcinoid by gene expression profiles.

Small cell lung cancer is a common type of lung cancer that is generally classified within the spectrum of neuroendocrine lung neoplasms. Using high-density cDNA arrays, we profiled gene expression of small cell lung cancers and compared these expression profiles to those of normal bronchial epithelial cells and pulmonary carcinoids, which are classified as benign neuroendocrine tumors. We found the overall expression profiles of two small cell lung cancer cell lines, two microdissected tissue samples of primary small cell lung cancer, and cultured bronchial epithelial cells to be relatively similar to one another, with an average Pearson correlation coefficient for these comparisons of 0.63. However, we found the expression profiles of small cell lung cancers (and bronchial epithelial cells) to be surprisingly dissimilar to those of two samples of pulmonary carcinoid tumors, with an average correlation coefficient for these comparisons of 0.20. We then compared the pulmonary carcinoid expression profiles to those of two samples of infiltrating astrocytic brain cancers (oligodendroglioma and high-grade astrocytoma) and found similarity of gene expression among these four samples (average correlation coefficient, 0.57). These gene expression profiles suggest that small cell lung cancers are closely related to (and possibly derived from) epithelial cells, and that pulmonary carcinoids are related to neural crest-derived brain tumors. More generally, our results suggest that broad profiles of gene expression may reveal similarities and differences between tumors that are not apparent by traditional morphological criteria.

The S387Y mutations of the transforming growth factor-beta receptor type I gene is uncommon in metastases of breast cancer and other common types of adenocarcinoma.

Recently, mutations of the transforming growth factor-beta receptor type I gene have been reported to occur at high frequency in breast cancer metastases, with all mutations being an identical C to A transversion at nucleotide 1160 of the gene (T. Chen et al, Cancer Res., 58: 4805-4810, 1998). This mutation would result in a serine to tyrosine substitution at codon 387 (S387Y) and would reportedly disrupt receptor function. Because this mutation reportedly occurred at high frequency in breast cancer metastases (42%) and much less frequently in primary breast cancer tumors (6%), this would seem to represent a pivotal genetic alteration in breast cancer progression. To further investigate the possible role of this specific genetic alteration in the progression of breast cancer and other forms of adenocarcinoma, we analyzed 20 breast cancer metastases, 15 lung adenocarcinoma metastases, and 13 colorectal cancer metastases for possible mutations at this site. Using both single-strand conformation polymorphism screening and sequencing, we found no mutations of this gene in any of our samples. Our results suggest the S387Y mutation of the transforming growth factor-beta receptor type I gene is not common in these types of human cancers.

Spreadsheet-based program for alignment of overlapping DNA sequences.

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.

Microsatellite instability is uncommon in breast cancer.

In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. We report here our experience with >10,000 amplifications of simple nucleotide repeats in noncoding genomic regions using DNA from 267 cases of breast cancer, including cases that represent all major histological types of breast cancer. We rarely (10 reactions) found unexpected bands in amplifications of tumor DNA that were not present in amplifications of normal DNA. Moreover, repeats of these reactions did not confirm microsatellite instability in a single case. We also evaluated the simple nucleotide repeats in the transforming growth factor type II receptor, insulin-like growth factor type II receptor, BAX, and E2F-4 genes, which are frequently mutated in tumors with microsatellite instability. No mutations of these genes were found in any of the 30 breast cancer cell lines and 61 primary breast cancer samples examined. These results indicate that mismatch repair errors characteristic of the MSI phenotype are uncommon in human breast cancer.

Breast development gives insights into breast disease.

AIMS: Studies of developing human breasts are essential for understanding the organogenesis as well as molecular pathogenesis of benign and malignant breast diseases. In this study we have examined the distribution of TGF-alpha, TGF-beta 1, tenascin-C and collagen type IV with the aim of starting to build a picture of the profile of molecules that may be involved in the development of the human breast. METHODS AND RESULTS: Ten fetal breasts (16 to 23 weeks of gestation) and 45 infant breasts, ranging in age from newborn to 2 years, were used in this study. Paraffin sections from these samples were immunostained with antibodies for these proteins and for Ki67 to elucidate the level of proliferative activity in different stages of breast development. TGF-alpha immunoreactivity was observed both in the stromal and the epithelial cells within fetal and infant breasts up to 25 days. TGF-beta 1 immunoreactivity was localized in the extracellular matrix. Tenascin-C was found around the neck of the developing breast bud and in the extracellular matrix of the infant with peaks in the newborn at 6-12 weeks. The immunoreactivity for type IV collagen was more intense in the region of the breast bud neck in the fetal breasts and reduced around the tips of lobular and terminal-end buds within the infant breasts. CONCLUSIONS: The distribution of the growth factors and extracellular matrix proteins within the developing human breast indicates that they play a significant role in different cellular compartments during morphogenesis and provides insights into breast disease.

The development of epithelial phenotypes in the human fetal and infant breast.

In order to explain the molecular events that contribute to benign and malignant breast disease, it is essential to understand the cellular context in which these are occurring. This study describes a detailed analysis of the epithelial phenotypes in the human fetal and infant breast and provides a starting point for such consideration. Using methacarn-fixed, paraffin sections from ten fetal and 45 infant breast, immunostained with a panel of antibodies to cytoskeletal proteins and kappa-casein, it has been possible to define in detail the chronological evolution of the major cell types in the human breast from 16 weeks of intrauterine life to 2 years of age, in both sexes. Cells at the tips of the lobular buds and terminal end buds have a characteristic cytoskeletal protein profile, suggesting that they may have the capacity to generate both basal cells and luminal cells. Based on the expression of cytoskeletal proteins in the developing fetal and infant breast, a model system has been proposed for mammary epithelial differentiation.

Allelic loss of chromosomal arm 8p in breast cancer progression.

Loss of heterozygosity (LOH) of chromosomal arm 8p has been reported to occur at high frequency for a number of common forms of human cancer, including breast cancer. The objectives of this study were to define the regions on this chromosomal arm that are likely to contain breast cancer tumor suppressor genes and to determine when loss of chromosomal arm 8p occurs during breast cancer progression. For mapping the tumor suppressor gene loci, we evaluated 60 cases of infiltrating ductal cancer for allelic loss using 14 microsatellite markers mapped to this chromosomal arm and found LOH of 8p in 36 (60%) of the tumors. Whereas most of these tumors had allelic loss at all informative markers, five tumors had partial loss of 8p affecting two nonoverlapping regions. LOH for all but one of the tumors with 8p loss involved the region between markers D8S560 and D8S518 at 8p21.3-p23.3, suggesting that this is the locus of a breast cancer tumor suppressor gene. We then studied LOH of 8p in 38 cases of ductal carcinoma in situ (DCIS) with multiple individually microdissected tumor foci evaluated for each case. LOH of 8p was found in 14 of the DCIS cases (36%), including 6 of 16 cases of low histological grade and 8 of 22 cases of intermediate or high histological grade. In four of these DCIS cases, 8p LOH was seen in some but not all of the multiple tumor foci examined. These data suggest that during the evolution of these tumors, LOH of 8p occurred after loss of other chromosomal arms that were lost in all tumor foci. Thus, LOH of 8p, particularly 8p21.3-p23, is a common genetic alteration in infiltrating and in situ breast cancer. Although 8p LOH is common even in low histological grade DCIS, this allelic loss often appears to be preceded by loss of other alleles in the evolution of breast cancer.

Homeobox genes: molecular link between congenital anomalies and cancer.

Homeobox-containing genes play a major role in the control of segmental identity during embryonic development in Drosophila. Abnormalities of these genes have been shown to produce a wide variety of congenital anomalies in invertebrates and in vertebrates. Many transgenic mice, which are mutant for homeobox genes, show a specific skeletal abnormality, similar to the human cervical rib. In humans, a relationship exists between malformations and tumours. Human cervical rib has been shown to be associated with an increased incidence of malignancy. Recent evidence indicates that homeobox genes might also play a role in carcinogenesis. In this article, we explore the possibility that alterations of homeobox genes might be the basic underlying aetiology for the association between congenital malformations and tumours, at least in a proportion of cases. We provide evidence in support of this argument and suggest areas of further research which would confirm this concept.

Expression of integrin subunits in the human infant breast correlates with morphogenesis and differentiation.

Integrins are widely expressed on normal tissues and their function is considered critical directly or indirectly with the control of cell growth and differentiation. Also, they are likely to play a crucial role in cell-matrix interactions during development. As the human breast develops after birth, it provides a rare opportunity in which to study human organogenesis. We have examined the distribution of integrins in the human infant breast with the aim of elucidating the possible role of these molecules in morphogenesis and differentiation. Necropsy breast specimens from six male and eight female infants, ranging in age from 1 day to 9 months, were used in this study. Cryostat sections were stained by the avidin-biotin complex technique, using a panel of monoclonal antibodies (MAbs) which recognize beta 1, alpha 2, alpha 6, beta 4, alpha v, and alpha v beta 3 integrin chains, which are candidate molecules for a role in mammory morphogenesis. MAbs to beta 1 (DH12) and alpha 2 (HAS3) showed positive membrane and cytoplasmic staining of basal cells and luminal epithelial cells. In addition, positive staining for the beta 1 integrin chain was found on fibroblasts. A MAb which recognizes the alpha 6 chain (MP4F10) showed positive staining of the basal cells and heterogeneous staining of the luminal epithelial cells, whilst beta 4 chain (439-9B) showed positive staining in the basement membrane domain of the basal cells with no staining of the luminal epithelial cells. There was a positive correlation between the intensity of expression and the structural development of the ductal system, with integrin expression reduced or absent in the end buds and lateral buds.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure and immunohistochemistry of the embryonic type of fat identified in the human infant breast.

In this paper we describe the light and electron microscopic appearance of the embryonic type of fat in human infant breast, together with immunocytochemical findings. This fat tissue was composed of numerous capillaries surrounded by a mixed population of undifferentiated mesenchymal cells and preadipocytes at various stages of differentiation. The preadipocytes were characterised by a number of cytoplasmic processes, varying numbers of lipid droplets, and an envelope of electrondense material outside the cell membrane. Immunocytochemistry showed a characteristic distribution of collagen type IV adjacent to and vimentin and S100 protein within the preadipocytes. This is the first report of the ultrastructure of the human mammary embryonic type of fat. The possible role of the embryonic type of fat in the development and growth of the human breast is discussed.

Expression of BCL-2 in primary breast cancer and its correlation with tumour phenotype. For the International (Ludwig) Breast Cancer Study Group.

BACKGROUND: The role of apoptosis (programmed cell death) in the development and progression of breast cancer is unknown. Recently the bcl-2 gene has been shown to block apoptosis and thus may promote tumour development. BCL-2 is localized to the luminal cells of the normal breast, which are considered to be the origin of malignant breast disease. PATIENTS AND METHODS: Immunocytochemistry using anti bcl-2- antibody was performed on 107 breast cancer specimens belonging to node-positive patients from the Ludwig Breast Cancer Studies I-IV and the results were correlated with survival, tumour grade, S-phase, oestrogen and progesterone receptor status and c-erb B-2 expression. Western and Southern blotting together with immunofluorescence were performed on the breast cancer cell lines BT-20, BT-474, MDA-MB-361, T47-D and MCF-7. RESULTS: In the breast cancer derived cell line MCF-7 BCL-2 is expressed to a level similar to that of the B-lymphoma cell line Karpas 231 with t(14;18)(q32.3;q21.3), but no evidence of a rearrangement or gene amplification was identified. In a study of 107 breast cancers from the International Breast Cancer Study Group Trials I-IV we have demonstrated a very significant inverse correlation of BCL-2 with c-erbB-2 expression (p = 0.002), and a positive correlation with oestrogen receptors (p = 0.001) and progesterone receptors (p = 0.05). In this study there was no correlation of expression with S-phase fraction in the tumours or with any stage in the cell cycle as assessed in MCF-7 cells. CONCLUSION: We conclude that BCL-2 might contribute to the malignant phenotype of breast cancer by modulation of biological behaviour of cancer cells.

Immunolocalisation of cell surface peptidases in the developing human breast.

The immunocytochemical distribution of three cell surface peptidases was investigated in samples of developing infant breast ranging in age from newborn to 9.5 months. We have previously demonstrated that in the adult breast these enzymes identify subpopulations of epithelial cells and fibroblasts. We therefore wished to address two questions: (a) At what stage in breast development can fibroblast subpopulations be identified, and (b) Is the distribution of these peptidases related to cellular differentiation and morphogenesis? At the histological level there was a cuff of stromal cells closely associated with the developing ductular and lobular structures. At all stages of ductular and lobular development the fibroblasts in this layer were consistently negative for dipeptidyl peptidase IV (DPP IV) and clearly distinguished from the fibroblasts in the surrounding matrix, some of which expressed DPP IV in an age-dependent manner. Within the infant breast aminopeptidase N (APN) was localised to luminal epithelial cells and all fibroblasts, whilst neutral endopeptidase (NEP) was specifically localised to myoepithelial cells. These results are considered in relation to the role of stromal-epithelial interactions during morphogenesis and the proposed function of these enzymes.