Microfluidic culture of single human embryonic stem cell colonies.

We have developed a miniaturized microfluidic culture system that allows experimentation on individual human embryonic stem cell (hESC) colonies in dynamic (flow applied) or static (without flow) conditions. The system consists of three inlet channels that converge into a cell-culture channel and provides the capability to spatially and temporally deliver specific treatments by using patterned laminar fluid flow to different parts of a single hESC colony. We show that microfluidic culture for 96 h with or without flow results in similar maintenance of hESC self-renewal, the capability to differentiate into three germ cell lineages, and to maintain a normal karyotype, as in standard culture dishes. Localized delivery of a fluorescent nucleic acid dye was achieved with laminar flow, producing staining only in nuclei of exposed cells. Likewise, cells in desired regions of colonies could be removed with enzymatic treatment and collected for analysis. Re-coating the enzyme treated area of the channel with extracellular matrix led to re-growth of hESC colonies into this region. Our study demonstrates the culture of hESCs in a microfluidic device that can deliver specific treatments to desired regions of a single colony. This miniaturized culture system allows in situ treatment and analysis with the ability to obtain cell samples from part of a colony without micromanipulation and to perform sensitive molecular analysis while permitting further growth of the hESC colony.

Association between the angiotensin-converting enzyme (insertion/deletion) and angiotensin II type 1 receptor (A1166C) polymorphisms and breast cancer among Brazilian women.

INTRODUCTION: We evaluated the association between components of the renin-angiotensin system and the development of breast cancer in a case-control study by means of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) and angiotensin II type 1 (AT( 1))-receptor A1166C polymorphisms. METHODS: Genotyping was performed by PCR-RFLP (restriction fragment length polymorphism) or PCR (polymerase chain reaction) using genomic DNA extracted from buccal cells of subjects with (101 cases) or without (307 controls) breast cancer. RESULTS: The frequencies of genotypes for ACE were: DD, ID and II (in %: cases: 60; 20; 20; controls: 46; 37; 17; p=0.019, chi(2)); and for AT(1)receptor were:AA,AC and CC (in %: cases: 65; 30; 5; controls: 51; 44; 5; p=0.114, chi( 2)).The results suggested that the A1166C polymorphism was not associated with breast cancer risk. On the other hand, for the ACE (I/D), there seemed to be different risks for cancer between cases and controls. CONCLUSIONS: The ID genotype was less frequently associated with the disease than were the DD or II; that is, women with the ID genotype were 3.1 times less likely to develop breast cancer than those with the other genotypes.The ID genotype might be protective against breast cancer and the ACE (I/D) polymorphism a possible target for developing genetic markers for breast cancer.

CD74-dependent deregulation of the tumor suppressor scribble in human epithelial and breast cancer cells.

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

Genetic polymorphisms of cytochrome P450cl7alpha (CYP17) and progesterone receptor genes (PROGINS) in the assessment of endometriosis risk.

We designed the present study in order to evaluate the eventual role of polymorphisms in the genes encoding cytochrome P450c17alpha (CYP17) and the progesterone receptor (PROGINS) as risk factors for endometriosis development. Eligible cases consisted of 121 women with surgically confirmed endometriosis who underwent treatment in a hospital in São Paulo, Brazil during the period from September 2003 to September 2005. The 281 controls were participants with normal gynecological as well as pelvic ultrasound evaluation, who did not have any gynecological conditions during their reproductive lives such as pelvic pain and/or dyspareunia nor infertility history. Genomic DNA was obtained from buccal cells and processed for DNA extraction using the GFX DNA extraction kit (GE Healthcare). The CYP17 (-34T-->C) polymerase chain reaction-restriction fragment length polymorphism assay has been described previously, as has the progesterone receptor polymorphism (PROGINS) detection assay. PROGINS heterozygosis genotype frequencies were shown to be statistically higher in endometriosis cases compared with controls. On the other hand, differences in the CYP17 polymorphism (-34T-->C) frequencies were not even close to significance (p = 0.278) according to our findings.