We introduce a beta-binomial sampling model that correctly incorporates both sources of variation. We show how to fit the parameters of this model, and introduce a test statistic for differential expression similar to a two-sample t-test.

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2,6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP r ) fibroblast variants were recovered at a median frequency of 12 ؋ 10 ؊5 from individual ears from progeny of crosses between mouse strains 129͞Sv and C3H͞HeJ. The frequency of DAP r variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development. Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP r variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT. Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo.Retinoblastoma is a prototype disease for understanding how loss of function of tumor suppressor genes (TSGs) leads to tumor formation. The so-called two-hit (two-mutational events) model explains elegantly the inheritance of genetic predisposition and development of retinal tumors (1). In familial cases, a preexisting RB1 germ-line mutation (the first hit) is inherited, predisposing the retinoblast cells to tumor development by requiring only a second mutational event (the second hit). In sporadic cases, somatic cells lack the predisposing mutation, and a retinoblast cell must acquire two separate RB1 mutations to progress to a tumor. In the two-hit model, the first hit, a rate-limiting step, renders a cell heterozygous or hemizygous at RB1. The second hit, which is frequently referred to as loss of heterozygosity (LOH), leads to the expression of the RB1 mutant phenotype (2). Because

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

The Pak4 serine/threonine kinase is highly expressed in many cancer cell lines and human tumors. While several studies have addressed the role for Pak4 in transformation of fibroblasts, most human cancers are epithelial in origin. Epithelial cancers are associated not only with changes in cell growth, but also with changes in the cellular organization within the three dimensional (3D) architecture of the affected tissues. Here we used immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the role for Pak4 in mammary tumorigenesis. iMMECs are an excellent model system for studying breast cancer they can grow in 3D-epithelial cell culture, where they form acinar structures that recapitulate in vivo mammary morphogenesis. While Pak4 is expressed at low levels in wild type iMMECs, it is overexpressed in response to oncogenes, such as oncogenic Ras and Her2/neu. Here we found that overexpression of Pak4 in iMMECs leads to changes in 3D acinar architecture that are consistent with oncogenic transformation. These include decreased central acinar cell death, abrogation of lumen formation, cell polarity alterations, and deregulation of acinar size and cell number. Furthermore, iMMECs overexpressing Pak4 form tumors when implanted into the fat pads of athymic mice. Our results suggest that overexpression of Pak4 triggers events that are important for the transformation of mammary epithelial cells. This is likely to be due to the ability of Pak4 to inhibit apoptosis and promote cell survival, and thus subsequent uncontrolled proliferation, and to its ability to deregulate cell shape and polarity.

Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain.

To develop a stable chronic obstructive pulmonary disease (COPD) model in rats. Sprague-Dawley rats were treated with cigarette-smoke inhalation (CSI) for 12 weeks, repetitive bacterial infection (RBI) for 8 weeks, or the combination of the two (CCR) for 12 weeks and followed up for the additional 20 weeks. Tidal volume (V T ), peak expiratory flow (PEF) and 50% V T expiratory flow (EF 50 ), histological changes in the lungs, and levels of the cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-10 in serum and bronchial alveolar lavage fluid (BALF) were examined at intervals during the 32 week study period. The right ventricular hypertrophy index (RVHI) was also determined at the same times. V T , PEF, and EF 50 were decreased in rats with COPD compared to the control. The expression of TNF-α, IL-8 and IL-10 increased in both serum and BALF with a similar trend. Bronchiole and arteriole wall thickness and the degree of bronchiole stenosis and alveolar size increased in COPD rats. RVHI was reduced gradually following the treatment. All of these changes were more pronounced in the CCR-treatment group than in the other groups. Our results have shown that CSI or RBI alone can induce COPD in rats, but that the combination of CSI with RBI induces a stable COPD that has more similarity to complications seen in patients with COPD. This combination may therefore provide a more appropriate model for study of human COPD.Key words pulmonary disease; chronic obstructive; animal model; pulmonary function; histological change; right ventricular hypertrophy index Chronic obstructive pulmonary disease (COPD) has been defined as a preventable and treatable pathologic condition characterized by partially reversible airflow limitation, 1) and is a major cause of morbidity and mortality throughout the world.2) Its prevalence among the Chinese population aged 40 or older is 8.2%.3) Development of COPD is slow and progressive in humans, with occasional exacerbations caused by an inflammatory response to triggering substances such as noxious gases, 1) bacteria 4-6) or viruses. [7][8][9][10][11] Four abnormalities are present in chronic, stable COPD: emphysema, small airway remodeling, pulmonary hypertension, and chronic bronchitis.Tobacco smoking and bacterial infection are the most common and important risk factors for COPD, 1,12,13) and they have each been used to establish animal COPD models. [14][15][16] Animal models have also used other noxious gases [17][18][19] and Pneumocystis carinii infection 20) in COPD induction. Short term induction protocols (days) 17,21) have resulted in a pulmonary inflammatory infiltrate, increased mucus production, and pulmonary edema. Long term induction protocols (weeks or months) 15,[17][18][19] have produced, in addition to the inflammatory infiltrate, emphysema and pulmonary remodeling characterized by fibrosis, and thickened bronchiole and arterial walls. Problems with animal models are that most of them are of short duration and the COPD produced does not correspond to the late...

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