Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

A high-throughput and sensitive method to measure global DNA methylation: application in lung cancer.

Background: Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets.

Methods: We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment.

Structure-based mutation analysis shows the importance of LRP5 beta-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling.

A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Wnts with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf 1 (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5-G171V) induces beta-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity.

Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer.

Background: We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy.

A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait.

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease.

Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter.

Background: Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations.

Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.

A novel human cystatin gene was identified in a differential display comparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer. Messenger RNAs from primary and metastatic tumor cells isolated from the same patient were compared. A partial cDNA was isolated that was expressed in the primary tumor cell line but not in the metastatic line.

A novel protease homolog differentially expressed in breast and ovarian cancer.

Background: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer.

Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells.

A gene encoding a protein related to the serpin family of protease inhibitors was identified as a candidate tumor suppressor gene that may play a role in human breast cancer. The gene product, called maspin, is expressed in normal mammary epithelial cells but not in most mammary carcinoma cell lines. Transfection of MDA-MB-435 mammary carcinoma cells with the maspin gene did not alter the cells' growth properties in vitro, but reduced the cells' ability to induce tumors and metastasize in nude mice and to invade through a basement membrane matrix in vitro.

Identification by differential display of alpha 6 integrin as a candidate tumor suppressor gene.

A new method of differential expression cloning called differential display (DD) has been used to screen for novel tumor suppressor genes involved in breast cancer. The screen is based on positive selection at the mRNA level for genes expressed in normal mammary epithelial cells but decreased or lost in corresponding tumor cells. A candidate tumor suppressor gene recovered by DD is integrin alpha-6 (alpha 6), a component of the heterodimeric integrin receptors alpha 6 beta 1 and alpha 6 beta 4.

Cytokine dysregulation in AIDS: in vivo overexpression of mRNA of tumor necrosis factor-alpha and its correlation with that of the inflammatory cytokine GRO.

The human immunodeficiency virus establishes an intimate interaction with the immune system. The virus can use cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (Il-1), to regulate its own expression by modifying the normal immunoregulatory network. We demonstrate that mRNA of the cytokine TNF-alpha from peripheral blood mononuclear cells is overexpressed in virtually all patients with AIDS who do not have active opportunistic infections compared with uninfected volunteers (p < 0.

An NF-kappa B-like transcription factor mediates IL-1/TNF-alpha induction of gro in human fibroblasts.

Normal human foreskin fibroblasts were used to examine transcriptional induction by IL-1 and TNF-alpha of the novel cytokine gro (melanoma growth-stimulating activity). Gro mRNA was expressed at levels 100-fold above background within 45 min of exposure to either IL-1 or TNF-alpha, in growing or serum-starved cells and a similar response was shown by IL-6. In contrast, as shown previously, gro mRNA was elevated only 10-fold by serum in starved but not in growing cells, similar to fos.

Identification of three related human GRO genes encoding cytokine functions.

The product of the human GRO gene is a cytokine with inflammatory and growth-regulatory properties; GRO is also called MGSA for melanoma growth-stimulatory activity. We have identified two additional genes, GRO beta and GRO gamma, that share 90% and 86% identity at the deduced amino acid level with the original GRO alpha isolate. One amino acid substitution of proline in GRO alpha by leucine in GRO beta and GRO gamma leads to a large predicted change in protein conformation.

High frequency of large spontaneous deletions of DNA in tumor-derived CHEF cells.

Spontaneous mutations arising at the HPRT locus were examined in 126 mutants recovered from a series of six CHEF-derived cell lines. Altered restriction fragment patterns were characterized by Southern blot hybridization, and gene expression by RNA blot hybridization. Point mutants and gene-expression mutants predominated in the control (nontumorigenic) 18-1D-3 cell line and in two tumor-derived lines, one of which (16-2 Tuk 4) displayed a mutator phenotype.

Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.

Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described.

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplified KRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization.

The KRAS gene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions.

Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells.

Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III.

Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.

The Chinese hamster embryo fibroblast cell line CHEF/18 is readily transfected by plasmid DNA. In the present transfection studies with CHEF/18 cells, focus formation induced by plasmids containing the mutant human c-Ha-ras gene EJ was compared with that of control plasmids without the EJ insert. The focus-forming activity of the transfected plasmid J132, a recombinant of the Harvey murine sarcoma virus LTR and the normal human c-Ha-ras1 in pBR322, also was assessed.

Resistance of human cells to tumorigenesis induced by cloned transforming genes.

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse.

Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.

5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells.

DNA transfer of focus- and tumor-forming ability into nontumorigenic CHEF cells.

CHEF/18 fibroblastic cells derived from a Chinese hamster embryo are diploid and nontumorigenic and require multiple steps of chemical treatment and selection to produce tumorigenic derivatives. In this report, CHEF/18 cells and a mutant capable of growing in medium with a low concentration of serum, LS1-1, were recipients in DNA transfer experiments using the calcium phosphate coprecipitation method. Focus formation with donor DNAs from tumor-derived CHEF cells and from human bladder carcinoma cell line EJ gave yields of 0.