Sagar Ghosh

Interleukin 6 secreted from adipose stromal cells promotes migration and invasion of breast cancer cells.

Excessive adiposity has long been associated with increased incidence of breast cancer in post-menopausal women, and with increased mortality from breast cancer, regardless of the menopausal status. Although adipose tissue-derived estrogen contributes to obesity-associated risk for estrogen receptor (ER)-positive breast cancer, the estrogen-independent impact of adipose tissue on tumor invasion and progression needs to be elucidated. Here, we show that adipose stromal cells (ASCs) significantly stimulate migration and invasion of ER-negative breast cancer cells in vitro and tumor invasion in a co-transplant xenograft mouse model. Our study also identifies cofilin-1, a known regulator of actin dynamics, as a determinant of the tumor-promoting activity of ASCs. The cofilin-1-dependent pathway affects the production of interleukin 6 (IL-6) in ASCs. Depletion of IL-6 from the ASC-conditioned medium abrogated the stimulatory effect of ASCs on the migration and invasion of breast tumor cells. Thus, our study uncovers a link between a cytoskeleton-based pathway in ASCs and the stromal impact on breast cancer cells.

Novel genetic mapping tools in plants

Abstract Use of DNA-based genetic markers [1] has forever changed the practice of genetics. In the 20 years since that discovery, many different types of DNA-based genetic markers have been used for the construction of genetic maps, for the analysis of genetic diversity, trait mapping, as well as for applied diagnostic purposes. A bewildering array of acronyms, such as RFLP, SSR, AFLP, RAPD, AP-PCR, DAF, SAMPL, and many others describes these methodologies [2] . AFLPs and SSRs have become especially popular due to the formers high multiplex ratio and the latters high degree of informativeness [3] . Also, arbitrary primer-based methods, such as RAPD, found their applications because of their simplicity. All of these methods constitute indirect approaches towards assessing DNA sequence differences: single nucleotide polymorphisms (SNPs, [4] ) and insertions/deletions (indels). A direct analysis of sequence difference between many individuals at a large number of loci has now become practical. Dramatic advances in sequencing technology have resulted in the determination of complete DNA sequences of many organisms including most notably human, and, from a plant scientists’ perspective, Arabidopsis [5] . The next important objective is to determine sequence diversity of genic and regulatory regions in these and other species. This would allow the understanding of the relationship between phenotypic diversity and genetic diversity. We discuss here the development and applications of SNP genetic markers in corn and other crop plants, and the contribution of these studies towards the understanding of the organization of genetic diversity in plants. We also discuss linkage disequilibrium-based trait mapping approaches.

Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression

Mutations in BRCA1 increase risks of familial breast and ovarian cancers, particularly among premenopausal women. While BRCA1 plays an active role in DNA repair, this function alone may not be sufficient to explain why BRCA1-associated tumors predominantly occur in estrogen-responsive tissues. Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Aromatase expression in ovarian granulosa cells dictates levels of circulating estrogen in premenopausal women, and its aberrant overexpression in breast adipose tissues promotes breast cancer growth. Here, we show that BRCA1 modulates aromatase expression in ovarian granulosa cells and primary preadipocytes. The cyclic AMP-dependent expression of aromatase in ovarian granulosa cells is inversely correlated with the protein level of BRCA1. Importantly, transient knockdown of BRCA1 enhances aromatase expression in both ovarian granulosa cells and primary preadipocytes. We propose that BRCA1 deficiency in epithelial and certain nonepithelial cells may result in combined effects of aberrant estrogen biosynthesis and compromised DNA repair capability, which in turn may lead to specific cancers in the breast and ovary.

Shiga toxin binds to activated platelets.

Summary.  Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)‐producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid‐citrate‐dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA‐washed preparations relative to ACD‐derived platelets. Binding of Stx was also increased with ACD‐derived platelets when activated with thrombin (1 U mL−1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA‐exposed platelets lost their normal discoid shape and were larger. P‐selectin (CD62P) exposure was significantly increased in EDTA‐washed preparations relative to ACD‐derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on ‘activated’ platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

Expression of Plasmodium falciparum C-terminal region of merozoite surface protein (PfMSP119), a potential malaria vaccine candidate, in tobacco

Abstract A gene encoding the C-terminal region of a major surface antigen of Plasmodium falciparum referred as PfMSP1 19 , a leading vaccine candidate for malaria, was cloned in plant transformation vector pBI121. Tobacco leaves were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin resistant plants were regenerated. The Pfmsp1 19 gene, in the transformed plants was identified by polymerase chain reaction amplification, Southern and northern hybridisation techniques. Expression of PfMSP1 19 protein was analysed by enzyme linked immunosorbent assay (ELISA) and Immunoblot assay, which indicated that transformed plants expressed MSP1 19 and displayed structural and immunological characteristics identical to the Escherichia coli expressed protein. This result is a step forward towards the development of an edible and low cost subunit vaccine against malaria, which is a fatal disease of the developing countries.

Deregulation of cofactor of BRCA1 expression in breast cancer cells

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four‐subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen‐dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer‐related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co‐depletion of the NELF subunits. In addition, our study suggests that a post‐transcriptional, proteasome‐independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis. J. Cell. Biochem. 103: 1798–1807, 2008.

Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element

Estrogen is critical to both normal mammary gland and breast cancer development. Circulating levels of estrogen in premenopausal women are primarily determined by the action of aromatase in ovarian granulosa cells that converts testosterone to estradiol. In the current study, we unraveled an important role of Jun proteins in modulating ovary-specific aromatase expression. Ectopic expression of the Jun proteins in a human granulosa cell line significantly inhibited an ovary-specific promoter (PII) of the aromatase gene, whereas expression of dominant-negative mutants of Jun led to increased promoter activity. The Jun-mediated repression was specific to the aromatase promoter, as Jun proteins stimulated known AP1-responsive promoters in the same cellular context. Both the activation and basic leucine zipper domains of Jun were required for the transcriptional repression. Electrophoretic gel mobility assay showed that endogenous Jun proteins bound to a functionally important cAMP-responsive element (CRE) in the PII promoter-proximal region. Alteration of the CRE-like site impaired both the cAMP-responsive transcriptional activation and Jun-mediated repression. Furthermore, chromatin immunoprecipitation indicated the presence of cJun at the proximal region of the native PII promoter. Taken together, our work suggests that Jun proteins may attenuate estrogen biosynthesis by directly downregulating transcription of the aromatase gene in ovarian granulosa cells.

An IL-6 link between obesity and cancer.

Obesity is a growing epidemic all over the world that by virtue of inducation of a chronic, low-grade, and systemic inflammation leads to an increased risk of a number of diseases, including cancer. IL-6 an important cytokine in the increased risk to cancer in obese patients mainly because of its pro-inflammatory activity. Some data suggest that IL-6 might increase the risk of certain cancers such as those that originate from breast, liver, prostate, colon, and esophagus. A better understanding of the regulation and role of IL-6 in obesity-associated cancer is required to develop effective therapeutic approaches.

Regulation of adipose oestrogen output by mechanical stress

Adipose stromal cells are the primary source of local oestrogens in adipose tissue, aberrant production of which promotes oestrogen receptor-positive breast cancer. Here we show that extracellular matrix compliance and cell contractility are two opposing determinants for oestrogen output of adipose stromal cells. Using synthetic extracellular matrix and elastomeric micropost arrays with tunable rigidity, we find that increasing matrix compliance induces transcription of aromatase, a rate-limiting enzyme in oestrogen biosynthesis. This mechanical cue is transduced sequentially by discoidin domain receptor 1, c-Jun N-terminal kinase 1, and phosphorylated JunB, which binds to and activates two breast cancer-associated aromatase promoters. In contrast, elevated cell contractility due to actin stress fibre formation dampens aromatase transcription. Mechanically stimulated stromal oestrogen production enhances oestrogen-dependent transcription in oestrogen receptor-positive tumour cells and promotes their growth. This novel mechanotransduction pathway underlies communications between extracellular matrix, stromal hormone output, and cancer cell growth within the same microenvironment.

Effects of insulin and oral anti-diabetic agents on glucose metabolism, vascular dysfunction and skeletal muscle inflammation in type 2 diabetic subjects†

To test potential differences between the actions of anti‐diabetic medications, we examined the effects of oral hypoglycaemic agents versus glargine–apidra insulin therapy in T2DM.