Scott M. Kahn

Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies.

Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori.

Sex hormone-binding globulin influences gene expression of LNCaP and MCF-7 cells in response to androgen and estrogen treatment.

Sex hormone-binding globulin (SHBG), a plasma protein that binds androgens and estrogens, also participates in the initial steps of a membrane-based steroid signaling pathway in human prostate and breast. We have recently shown that SHBG is expressed at the mRNA and protein levels in the prostate and breast. In this study, we addressed whether locally expressed SHBG: (1) Functions to regulate activation of membrane-based steroid signaling and (2) influences activation of the androgen (AR) and estrogen (ER) receptors. Using microarray analysis, we identified specific genes that are influenced by SHBG expression in LNCaP and MCF-7 cells in a manner consistent with each of these properties. These findings suggest that locally expressed SHBG can play a functional role in the steroid responsiveness of prostate and breast cells through multiple signaling pathways and that perturbations in local SHBG expression could contribute to prostate and breast cancer.

Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

BackgroundHuman sex hormone-binding globulin (SHBG) regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (PL). The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (PT), and two minor transcripts.ResultsWe report that transcriptional expression of the human SHBG gene is far more complex than previously described. PL and PT direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of PL and PT, and present evidence for a third SHBG gene promoter (PN) within the neighboring FXR2 gene; PN regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N). Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. PL- PT- and PN-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells.ConclusionThese results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

The Role of Protein Kinase C in Signal Transduction, Growth Control and Lipid Metabolism

Of the numerous protein kinases present in mammalian cells protein kinase C (PKC) is of particular interest to the field of lipid metabolism because various lipids influence the activation of this enzyme system. These lipid activators include phosphatidylserine, cis-unsaturated fatty acids (arachidonic acid, oleic acid), diacylglycerol, inositol phosphates, fecapentaene, phorbol ester tumor promoters and related compounds, and cholesterol sulfate. The activation of PKC can, in turn influence lipid metabolism by causing feedback inhibition of phospholipase C., activation of phospholipase A2 and activation of phospholipase D (1–6).

Immunohistochemical and In-Situ Detection of Sex Hormone-binding Globulin (SHBG) Expression in Breast and Prostate Cancer: Implications for Hormone Regulation

Sex Hormone Binding Globulin (SHBG), in addition to regulating free concentrations of steroid sex hormones in plasma, also participates in membrane-based steroid signaling in breast and prostate cells. In this study, we address whether the breast and prostate can synthesize their own SHBG. SHBG mRNA and protein were detected in human breast and prostate cancer cell lines, as well as in normal and cancerous breast and prostate tissue where it was prominent in epithelial cells. In cultured human LNCaP prostate cancer cells, SHBG was found both in the cytoplasm, and on the outer cell membrane where it was presumably bound to its high affinity receptor (RSHBG). When LNCaP cells were treated with 2-methoxyestradiol (2MeOE2), an antagonist of SHBG binding to RSHBG, membrane-associated SHBG was eliminated. These results demonstrate that the breast and prostate can synthesize SHBG locally, and that SHBG can bind to, and perhaps participate in autocrine and/or paracrine signaling through RSHBG. Therefore, perturbations in SHBG expression in breast and prostate tumors may affect the regulation of steroid signaling through RSHBG and target SHBG-mediated biologic properties at the cellular level.

SEX HORMONE BINDING GLOBULIN AND STEROID SIGNALING AT THE CELL MEMBRANE

Sex hormone-binding globulin (SHBG) both regulates the free concentration of certain steroids in plasma and participates in a cellular steroid signaling pathway originating at the cell membrane. The biochemistry of SHBG-mediated steroid signaling has been described in some detail. It is clear that it is a system that is distinct from steroid signaling through classical nuclear steroid hormone receptors. We will summarize the initial steps of steroid signaling through SHBG and discuss biologic events that are attributed to, or are speculated to have SHBG-mediated steroid signaling as their basis. Finally, we talk about the local expression of SHBG in target tissues and its implications for the regulation of cellular steroid signaling.

Sex Hormone-Binding Globulin as a Modulator of the Prostate “Androgenome”

Sex hormone-binding globulin (SHBG) is a sex steroid binding protein, originally described in humans as the major binding protein for estrogens and androgens in plasma (Anderson, 1974; Avvakumov, et al, 2010). By governing equilibrium conditions in plasma between bound and free sex steroids, SHBG regulates the availability of the latter to hormonally responsive tissues. Along with regulating free steroid concentrations in plasma, it is increasingly evident that SHBG also participates in other biological processes. These include, but are not limited toactivation of a rapid, membrane based steroid signaling pathway in tissues such as the prostate and breast (Rosner et al, 2010); spermatogenesis (Selva and Hammond, 2006); and a yet to be determined consequence of co-localization with oxytosin in brain cells (Caldwell et al, 2006). Plasma based SHBG is extensively studied, especially in the context of its regulation of free steroid concentrations and epidemiologic associations. The origin of plasma SHBG is, for all intents and purposes, the liver (Khan et al, 1981; Pugeat et al, 2010) (a differentially glycosylated isoform, androgen binding protein (ABP) is synthesized in the testis (Vigersky et al, 1976)). However, we now know that SHBG is also synthesized, albeit to a much lesser degree, in certain hormonally responsive tissues (Kahn et al, 2002). Early studies demonstrated immunoreactive SHBG in the prostate and breast (Bordin & Petra 1980; Tardivel-Lacombe et al, 1984; Sinnecker et al, 1988; 1990; Meyer et al, 1994; Germain et al, 1997), though its origin (local synthesis vs. import from plasma) was unclear. Other studies demonstrated SHBG mRNA in certain nonhepatic tissues (Larrea et al, 1993; Misao et al, 1994; 1997; Moore et al, 1996; Murayama et al, 1999), and one reported both SHBG protein and mRNA together in fallopian tube tissue (Noe, 1999). In 2002, we reported that human prostate tissue expresses both SHBG mRNA and protein, as do prostate cancer cell lines (Hryb et al, 2002), suggesting that SHBG is indeed locally

I. Bernard Weinstein: In Memoriam (1930–2008)

On November 3, 2008, we lost a true gentleman and visionary with the passing of Dr. I. Bernard Weinstein. Bernie was a dear friend and distinguished colleague to many in the scientific community. And to those fortunate enough to be included among Bernies “second family” of students, postdocs,

Studies on the Role of Protein Kinase C in Multistage Carcinogenesis and Their Relevance to Risk Extrapolation

One of the most intensively studied non-genotoxic carcinogens is the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Since TPA appears to exert its biologic effects through protein kinase C (PKC), a key enzyme in signal transduction, we have studied this enzyme in considerable detail. To explore the role of PKC in carcinogenesis we developed various types of cell lines that overproduce the PKC βI isoform. To gain further insight into the relationship between PKC structure and its function in tumor promotion we expressed deletion mutants of PKC βI in a rat fibroblast cell line. These studies provide genetic evidence that PKC plays a critical role in growth control and the action of certain growth factors, tumor promoters and oncogenes. The findings are discussed within the context of multistage carcinogenesis, risk assessment, and cancer prevention.