MDCK Cystogenesis Driven by Cell Stabilization within Computational Analogues

University of California San Diego, United States of America
PLoS Computational Biology (Impact Factor: 4.62). 04/2011; 7(4):e1002030. DOI: 10.1371/journal.pcbi.1002030
Source: PubMed


The study of epithelial morphogenesis is fundamental to increasing our understanding of organ function and disease. Great progress has been made through study of culture systems such as Madin-Darby canine kidney (MDCK) cells, but many aspects of even simple morphogenesis remain unclear. For example, are specific cell actions tightly coupled to the characteristics of the cell's environment or are they more often cell state dependent? How does the single lumen, single cell layer cyst consistently emerge from a variety of cell actions? To improve insight, we instantiated in silico analogues that used hypothesized cell behavior mechanisms to mimic MDCK cystogenesis. We tested them through in vitro experimentation and quantitative validation. We observed novel growth patterns, including a cell behavior shift that began around day five of growth. We created agent-oriented analogues that used the cellular Potts model along with an Iterative Refinement protocol. Following several refinements, we achieved a degree of validation for two separate mechanisms. Both survived falsification and achieved prespecified measures of similarity to cell culture properties. In silico components and mechanisms mapped to in vitro counterparts. In silico, the axis of cell division significantly affects lumen number without changing cell number or cyst size. Reducing the amount of in silico luminal cell death had limited effect on cystogenesis. Simulations provide an observable theory for cystogenesis based on hypothesized, cell-level operating principles.

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    • "The biological referents for demonstrations are simple cell culture cysts having an inner layer of luminal epithelial cells enclosing a lumen and an outer layer of myoepithelial cells. Discussion of these cell types and structures is beyond the scope of this paper and can be found elsewhere [47, 48].Figure 5 is an illustration of an idealized 2D cyst showing the basic layout for 2D V-cells, and Additional file 3 provides a video of cysts which shows movement and rotation [49] . Demonstrations employ three components . "

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    • "Felodipine disposition characteristics are different, but the basic processes and general, subject physiology are assumed to be similar between the two studies, which allowed us to begin by adopting the earlier analog. We then followed the iterative refinement protocol to extend the analog phenotype and achieve the new set of validation targets without having to reengineer the whole system, and without compromising already validated features and behaviors [13], [14]. The protocol starts with specifying referent attributes to be targeted, e.g., drug and metabolite concentration-time data, histological and biochemical measurements, morphological characteristics, etc. Next, an initial (small) subset of attributes is selected, and an analog is constructed, tested, and revised iteratively until the analog exhibits the targeted attributes within a prespecified level of similarity, thereby achieving a level of validation. "
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    • "It is noteworthy that tight regulation of cyst size, shape and polarization is critical for normal kidney development and functions. Disruption of these regulatory mechanisms leads to an array of diseases including autosomal dominant polycystic kidney disease, stenosis, and cancer [37]. Our previous data [32] and current studies showed that in 3-D culture, p53 knockdown alone is unable to alter MDCK cell morphology, although the cells display enhanced proliferation and migration activities. "
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    ABSTRACT: Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H) or R261H (human equivalent R273H) in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors (Snail-1, Slug and Twist) is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.
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