Elizabeth Herring

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Integrin-linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin-dependent mechanism

Integrin‐linked kinase (ILK) plays a role in integrin signaling‐mediated extracellular matrix (ECM)–cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold‐function interacting partners, PINCH‐1, α‐parvin, and β‐parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non‐proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH‐1, α‐parvin, and β‐parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown‐induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt–villus axis homeostasis—especially with regard to basement membrane fibronectin deposition—as well as cell proliferation, spreading, and migration. J. Cell. Physiol. 222: 387–400, 2010.

Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity

BackgroundIntegrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking.MethodsIn this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line.ResultsUsing variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc.ConclusionThe findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.

Laminins in the developing and adult human small intestine: relation with the functional absorptive unit.

The expression of the five laminin α‐chains was analyzed in the developing and mature human small intestine at the protein and transcript levels in order to further delineate specific involvement of individual laminins in relation to the epithelial cell state as defined along the functional crypt‐villus axis. The results show that all of the α‐laminin transcripts are expressed in significant amounts in the small intestine relative to a panel of other tissues and organs. Further analysis of their expression by indirect immunofluorescence and semi‐quantitative and quantitative RT‐PCR demonstrates a close correlation between transcript and protein expression, distinct epithelial and mesenchymal origins, as well as differential occurrence in intestinal basement membranes according to developmental stage, along the crypt‐villus axis and in compartment‐related experimental intestinal cell models. Taken together, the data point out the prime importance of α2‐, α3‐, and α5‐containing laminins for the development and maintenance of the functional human intestinal epithelium. Developmental Dynamics 236:1980–1990, 2007.

Differential expression of the integrins α6Aβ4 and α6Bβ4 along the crypt–villus axis in the human small intestine

The integrin α6 subunit exists as two different variants, termed α6A and α6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin α6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt–villus axis. Indeed, proliferative cells of the crypt were found to predominantly express α6A, while differentiated enterocytes and Paneth cells expressed the α6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two α6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the α6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.

A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome.

Abstract Secondary monolayer cultures from whole mouse embryos were infected at 39° with ts -P155, an early temperature-sensitive mutant of polyoma virus that transforms but replicates poorly at this temperature ( W. Eckhart, 1969 , Virology 38 , 120–125; W. Eckhart, 1974 , Cold Spring Harbor Symp. Quant. Biol. 39 , 37–40). The cultures were then passaged at 39° in the presence of polyoma virus antiserum until sufficient cell degeneration occurred to allow detection of foci of cells exhibiting lack of contact-inhibition. From one such focus, a cell strain—designated Cyp—was derived and cloned at 39°. Whether cloned or uncloned, Cyp cells all seem to exhibit essentially the same phenotype. At 39°, they display several properties of transformed cells and, as indicated by immunofluorescence, synthesize polyoma T antigen. They do not produce any readily detectable amounts of viral DNA or V antigen and only low amounts of viral hemagglutinin at 39°, although they are still fully permissive for polyoma virus replication at this temperature. Upon transfer to 33°, Cyp cells undergo a massive cytopathic effect detectable within 30 to 34 hr and produce large amounts of infectious, temperature-sensitive polyoma virus. Viral DNA accumulating in Cyp cells after temperature shift-down consists primarily of covalently closed monomers and, upon cleavage by Hpa II, yields fragments characteristic of the polyoma virus strain from which the ts -P155 mutant had been derived. When present in the medium during temperature shift-down, polyoma virus antiserum neither curtails the cytopathic effect nor depresses viral DNA synthesis, although it suppresses virus production. These observations are in agreement with the results of infectious center assays, suggesting that most Cyp cells produce virus following transfer to 33°.

Droplet digital PCR for quantification of ITGA6 in a stool mRNA assay for the detection of colorectal cancers

AIM To investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening. METHODS ddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods. ITGA6 and ITGA6A were chosen for this proof-of-concept study based on their relative medium and low abundance in stool samples, respectively, as established in a previous study. RESULTS We found that the ddPCR and qPCR methods performed equally well in this TaqMan duplex assay for the detection of ITGA6 and ITGA6A transcripts in stools of patients with colorectal lesions. For ITGA6, receiver operating characteristic (ROC) curve analysis showed comparable areas under the curve of 0.91 (P < 0.0001) and 0.89-0.90 (P < 0.0001) for the prediction of advanced adenomas and colorectal cancers, respectively. ITGA6A, which was detected at very low levels in control patients, was found to be significantly elevated (over 40 times) in stage II and III colorectal cancers (P < 0.0002). Comparison of the two sets of data revealed a strong correlation of the copy numbers obtained by ddPCR and qPCR for both ITGA6 and ITGA6A. CONCLUSION We found that ITGA6 and ITGA6A detection in stools of patients with colorectal cancers with ddPCR is comparable to that of qPCR using TaqMan assays.

Use of integrin alpha 6 transcripts in a stool mRNA assay for the detection of colorectal cancers at curable stages

Objective An important criterion for colorectal cancer (CRC) screening is the ability to detect lesions at a curable stage. In the present study, we have assessed the integrin α6 subunit transcript (ITGA6) as part of a stool assay for the detection of colorectal lesions. Results In comparison with control samples, ITGA6 levels were found to be significantly increased at all stages (P < 0.01). Receiver operating characteristic analysis revealed areas under the curve of 0.89 for the prediction of CRC with 81% sensitivity and 88% specificity and of 0.90 for the prediction of advanced adenomas (Ad) with 75% sensitivity and 88% specificity. The ITGA6A variant was also found to be increased relative to ITGA6 in stage II and III CRCs. Combining ITGA6 with other selected transcripts and/or immunochemical fecal occult blood test (iFOBT) results further increased sensitivity and specificity for the detection of colorectal lesions. Patients and Methods ITGA6 detection used alone and under various combinations including detection of other mRNA markers and iFOBT was assessed on stool samples obtained from 175 patients (91 CRCs, 24 Ad and 60 healthy controls). Conclusions These data confirm the usefulness and reliability of an mRNA stool assay for the detection of colorectal lesions. The validation of additional candidate genes and their analysis in multiplex qPCR represents a powerful and robust approach that can be combined with iFOBT results to improve the detection of colorectal lesions.

Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells.

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the beta-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.

A Stool Multitarget mRNA Assay for the Detection of Colorectal Neoplasms

Noninvasive screening methods for the detection of colorectal cancers (CRC) at curable stages rely on the identification of specific biomarkers. Our group has shown that mRNA stool assays represent a powerful and robust approach for the prediction of colorectal neoplasms. In this methodological chapter, we describe the procedures to isolate good quality stool RNA and the steps to evaluate the levels of specific host mRNA markers such as ITGA6, MYC, and GADD45B using TaqMan-based quantitative and droplet digital PCR approaches.