Mariano J. Scian

MicroRNAs as Biomarkers in Solid Organ Transplantation

Important progress has been made in improving short‐term outcomes in solid organ transplantation. However, long‐term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response. MicroRNAs (miRNAs) are a class of small single‐stranded noncoding RNAs that function through translational repression of specific target mRNAs. MiRNA expression has been associated with different diseases and physiological conditions. Moreover, miRNAs have been detected in different biological fluids and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA detection, their disease specificity and the availability of accurate techniques for detecting and monitoring these molecules has encouraged a pursuit of miRNA biomarker research and the evaluation of specific applications in the transplant field. miRNA expression might develop as excellent biomarkers of allograft injury and function. In this minireview, we summarize the main accomplishments of recently published reports focused on the identification of miRNAs as biomarkers in organ quality, ischemia‐reperfusion injury, acute rejection, tolerance and chronic allograft dysfunction emphasizing their mechanistic and clinical potential applications and describing their methodological limitations.

Tumor-Derived p53 Mutants Induce NF-κB2 Gene Expression

ABSTRACT Overexpression of mutant p53 is a common theme in tumors, suggesting a selective pressure for p53 mutation in cancer development and progression. To determine how mutant p53 expression may lead to survival advantage in human cancer cells, we generated stable cell lines expressing p53 mutants p53-R175H, -R273H, and -D281G by use of p53-null human H1299 (lung carcinoma) cells. Compared to vector-transfected cells, H1299 cells expressing mutant p53 showed a survival advantage when treated with etoposide, a common chemotherapeutic agent; however, cells expressing the transactivation-deficient triple mutant p53-D281G (L22Q/W23S) had significantly lower resistance to etoposide. Gene expression profiling of cells expressing transcriptionally active mutant p53 proteins revealed the striking pattern that all three p53 mutants induced expression of approximately 100 genes involved in cell growth, survival, and adhesion. The gene NF-κB2 is a prominent member of this group, whose overexpression in H1299 cells also leads to chemoresistance. Treatment of H1299 cells expressing p53-R175H with small interfering RNA specific for NF-κB2 made these cells more sensitive to etoposide. We have also observed activation of the NF-κB2 pathway in mutant p53-expressing cells. Thus, one possible pathway through which mutants of p53 may induce loss of drug sensitivity is via the NF-κB2 pathway.

Modulation of Gene Expression by Tumor-Derived p53 Mutants

p53 mutants with a single amino acid substitution are overexpressed in a majority of human cancers containing a p53 mutation. Overexpression of the mutant protein suggests that there is a selection pressure on the cell indicative of an active functional role for mutant p53. Indeed, H1299 cells expressing mutant p53-R175H, p53-R273H or p53-D281G grow at a faster rate compared with a control cell line. Using p53-specific small interfering RNA, we show that the growth rate of mutant p53-expressing cells decreases as mutant p53 level decreases, demonstrating that the increased cellular growth is dependent on p53 expression. Increased growth rate is not observed for H1299 cell clones expressing mutant p53-D281G (L22Q/W23S), which has been shown to be defective in transactivation in transient transcriptional assays. This shows that the increased growth rate imparted by mutant p53 in H1299 cells requires the transactivation function of mutant p53. By performing microarray hybridization analyses, we show that constitutive expression of three common p53 mutants (p53-R175H, p53-R273H, and p53-D281G) in H1299 human lung carcinoma cells evokes regulation of a common set of genes, a significant number of which are involved in cell growth regulation. Predictably, H1299 cells expressing p53-D281G (L22Q/W23S) are defective in up-regulating a number of these genes. The differences in expression profiles induced by individual p53 mutants in the cells may be representative of the p53 mutants and how they can affect gene expression resulting in the observed “gain of function” phenotypes (i.e., increased growth rate, decreased sensitivity to chemotherapeutic agents, and so forth).

Tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes

We have studied the mechanism of mutant p53-mediated oncogenesis using several tumor-derived mutants. Using a colony formation assay, we found that the majority of the mutants increased the number of colonies formed compared to the vector. Expression of tumor-derived p53 mutants increases the rate of cell growth, suggesting that the p53 mutants have ‘gain of function’ properties. We have studied the gene expression profile of cells expressing tumor-derived p53-D281G to identify genes transactivated by mutant p53. We report the transactivation of two genes, asparagine synthetase and human telomerase reverse transcriptase. Quantitative real-time PCR confirms this upregulation. Transient transfection promoter assays verify that tumor-derived p53 mutants transactivate these promoters significantly. An electrophoretic mobility shift assay shows that tumor-derived p53-mutants cannot bind to the wild-type p53 consensus sequence. The results presented here provide some evidence of a possible mechanism for mutant p53-mediated transactivation.

Wild-type p53 and p73 negatively regulate expression of proliferation related genes

When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or β-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21.

The urine microRNA profile may help monitor post-transplant renal graft function.

Non-invasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation and the longitudinal validation studies for non-invasive monitoring of graft function. Of 1,733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time-points post-kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early post-kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.

Characterization of a Whole Smoke In Vitro Exposure System (Burghart Mimic Smoker-01)

In vitro systems are frequently used to study mechanisms of mainstream cigarette smoke (MS)-induced lung injury. Traditional methods of exposure involve the capture of MS particulate phase with filter pads or bubbling MS through phosphate buffered saline (PBS) or cell culture medium. Although useful for in vitro experiments, these exposure methods may fail to capture potential interactions between the gas and particulate phases. To better understand the effect of MS on the human airway, in vitro whole smoke exposure systems that utilize freshly generated whole smoke are needed. Here we report the characterization of a new in vitro whole smoke exposure system (Burghart Mimic Smoker-01 (MSB-01)). This system uses a smoke distribution manifold to simultaneously deliver MS to each well of a 96-well plate. Intraday and interday variations for particulate matter deposition were less than 5% and 13% respectively. Cytotoxicity measurements using lung epithelial BEAS-2B cells indicate variations in calculated EC50 (half maximal effective concentration) values of 13% intraday and 20% interday. Smoke particulate losses and changes in particle size distribution were also analyzed. The data indicate that 45–50% of the MS generated at the smoking ports is lost within the system prior to delivery into the exposure chamber; however, no changes in particle size distribution were detected throughout the system. Overall, the MSB-01 reproducibly delivered mainstream cigarette smoke in a dose dependent manner across the multiwell plate. The MSB-01 is a high throughput system capable of exposing cells to both the MS particulate and gas/vapor phases simultaneously.

Gene Expression Changes Are Associated With Loss of Kidney Graft Function and Interstitial Fibrosis and Tubular Atrophy: Diagnosis Versus Prediction

Background. Loss of kidney graft function due to interstitial fibrosis (IF) and tubular atrophy (TA) is the most common cause of kidney allograft loss. Methods. One hundred one allograft tissues (26 samples with IF/TA, 17 normal allografts, and an independent biopsy group collected at 3 month [n=34] posttransplantation) underwent microarray analysis to identify early detection/diagnostic biomarkers of IF/TA. Profiling of 24 allograft biopsies collected at or after 9-month posttransplantation (range 9–18 months) was used for validation. Three-month posttransplantation biopsies were classified as IF/TA nonprogressors (group 1) or progressors (group 2) using graft function and histology at 9-month posttransplantation. Results. We identified 2223 differentially expressed probe sets between IF/TA and normal allograft biopsies using a Bonferroni correction. Genes up-regulated in IF/TA were primarily involved in pathways related to T-cell activation, natural killer cell-mediated cytotoxicity, and programmed cell death. A least absolute shrinkage and selection operator model was derived from the differentially expressed probe sets, resulting in a final model that included 10 probe sets and had 100% training set accuracy. The N-fold crossvalidated error was 2.4% (sensitivity 95.8% and specificity 100%). When 3-month biopsies were tested using the model, all the samples were classified as normal. However, evaluating gene expression of the 3-month biopsies and fitting a new penalized model, 100% sensitivity was observed in classifying the samples as group1 or 2. This model was evaluated in the sample set collected at or after 9-month posttransplantation. Conclusions. An IF/TA gene expression signature was identified, and it was useful for diagnosis but not prediction. However, gene expression profiles at 3 months might predict IF/TA progression.

Hetero-oligomerization does not compromise ‘gain of function’ of tumor-derived p53 mutants

Tumor-derived p53 mutants activate transcription from promoters of various growth-related genes. We tested whether this transactivation function of the mutant protein is sufficient to induce tumorigenesis (‘gain of function’). Tumor-derived mutant p53-281G transactivates the promoters of human epidermal growth factor receptor (EGFR) and human multiple drug resistance gene (MDR-1). To determine whether the C-terminal domain functions only as an oligomerization domain in mutant p53-mediated transactivation, we have replaced the tetramerization domain of p53 by a heterologous tetramerization domain; although this mutant protein formed tetramers in solution, it failed to transactivate significantly. Therefore, for successful mutant p53-mediated transactivation, sequences near the C-terminus of mutant p53 are required to perform functions in addition to tetramerization. We also demonstrate that co-expression of a deletion mutant of p53 (p53 del 1-293), which retains the p53 oligomerization domain, inhibits this transactivation. p53 del 1-293 co-immunoprecipitates with p53-281G suggesting that hetero-oligomers of p53-281G and p53 del 1-293 are defective in transactivation. We also show that a cell line stably transfected with p53-281G expresses higher levels of endogenous NF-κB and proliferating cell nuclear antigen (PCNA) compared to that transfected with vector alone. On co-expression, p53 del 1-293 lowered the levels of NF-κB and PCNA in p53-281G-expressing cells. However, on co-expression, p53 del 1-293 did not inhibit the tumorigenicity and colony forming ability of p53-281G expressing cells. Our earlier work showed that a deletion of the C-terminal sequences of p53-281G overlapping the oligomerization domain obliterates ‘gain of function’. Taken together, the above information suggests that the C-terminal sequences have some critical role in ‘gain of function’ in addition to transactivation.

Chemical analysis of cigarette smoke particulate generated in the MSB-01 in vitro whole smoke exposure system

Cigarette mainstream smoke (MS) is a dynamic aerosol consisting of a gas–vapor phase and a particulate phase. In recent years, novel in vitro whole smoke exposure systems have been developed to expose cells directly to whole MS. One such system is the Burghart Mimic Smoker-01 (MSB-01). Our previous data using the MSB-01 indicated that a 50 ± 10% loss of particulate matter occurred prior to MS delivery into the exposure chamber. Additionally, a change in aerosol particle diameter was also measured, suggesting that the chemical composition of MS might be changing within the system. In this study, we have expanded on our previous work and compared the particulate phase chemical composition of undiluted and diluted MS generated by the instrument and that of the MS delivered into the exposure chamber. The average percent delivery of cigarette smoke condensate (CSC) detected for all the measured chemical constituents was 35 ± 13% for undiluted MS and 23 ± 8% for 1:1 diluted MS. The data also indicate that under our experimental conditions, incomplete mixing of the freshly generated MS occurs during its dilution by the system. Taken together, the data presented here show that significant chemical changes occur between the generation of MS by the system and its delivery into the exposure chamber. This indicates that due to the dynamic nature of cigarette smoke, it is important to characterize the exposure conditions in order to gain the best insight and accurately correlate exposure with biological endpoints.