Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

The regulation of JAB1 and its role in herceptin resistance

The Jun activation domain-binding protein (JAB1) is a c-Jun co-activator and a member of the COP9 signalosome. Additionally, it has recently been named a key negative regulator of the cyclin-dependent kinase inhibitor, p27. JAB1 overexpression has been observed in breast cancer and correlates with low p27 levels as well as poor prognosis, yet the mechanism of JAB1 deregulation is unknown. Data from our laboratory suggest that constitutive transcriptional activation of the jab1 gene is responsible for JAB1 protein overexpression. Therefore, we hypothesized that overexpression of JAB1 in breast cancer can be attributed to increased transcriptional activity. To identify potential positive regulators of JAB1, we characterized the promoter and found a 128 bp region that was critical for jab1 transcriptional activation. Our studies show that two oncogenic transcription factors, C/EBPβ and STAT3, play an important role in modulating jab1 transcription. Further, we have identified jab1 as a direct target gene of the SRC/STAT3 pathway. These studies provide insight to the mechanism of JAB1 overexpression in breast cancer and open up possibilities for therapies to inhibit its expression. ^ The development of the humanized monoclonal antibody, Herceptin (trastuzumab) targeting the HER2 (ErbB2) receptor has provided promising treatment to patients with aggressive HER2 positive breast cancer. However, many patients are resistant to Herceptin and additional therapies are needed to overcome resistance. Recent findings indicate that one mechanism of resistance involves AKT phosphorylation and subsequent mislocalization of the cyclin dependent kinase inhibitor, p27. We examined whether JAB1 facilitated degradation of p27 may be another mechanism of resistance to Herceptin. Our studies show that overexpression of JAB1 inhibited Herceptin induced G1-arrest and p27 accumulation. Interestingly, increased JAB1 levels were observed in two BT-474 Herceptin resistant clones. Targeted silencing of JAB1 increased p27 protein levels, reinstated a G1 checkpoint, and reduced cellular proliferation in the resistant clones. Our studies have demonstrated that inhibition of JAB1 sensitizes Herceptin resistant cells to treatment. Therefore, inhibition of JAB1 could provide a novel method of sensitizing resistant tumors to Herceptin-induced tumor growth arrest. ^

Carcinoma of the lower uterine segment: A newly described association with Lynch Syndrome

Purpose. We performed a case-comparison study to describe the characteristics of LUS tumors and their association with risk factors for endometrial cancer. ^ Patients and Methods. From January 1996 through October 2007, 3,892 women were identified with a diagnosis of primary endometrial carcinoma or primary cervical adenocarcinoma. Pathology records from the 1,009 women who had a hysterectomy were reviewed. Subjects were included in the LUS group only if the tumor was clearly originating from the area between the lower corpus and upper cervix in the hysterectomy specimen. The LUS group was compared to all patients with endometrial corpus carcinoma who underwent hysterectomy at our institution in a 12-month period randomly selected from the study period. Risk factors for endometrial carcinoma such as body mass index (BMI) and Lynch Syndrome were assessed. Expression of estrogen receptor (ER), vimentin, carcinoembryonic antigen (CEA), p16, and human papilloma virus DNA (HPV DNA) was assessed; this panel is known to be effective in distinguishing adenocarcinomas of endometrial versus endocervical origin. Fisher's Exact, Chi-square, Mann-Whitney, and Student's t-tests were utilized for statistical analysis. ^ Results. Thirty-five of 1,009 women had endometrial carcinoma of the LUS (3.5%; 95% CI: 2–4%). Compared to patients with corpus tumors, LUS patients were younger (54.2 vs. 62.9 years, P = .001), had higher stage (P < .001), and more invasive tumors (P = .001). Preoperative diagnosis of the LUS tumors more frequently included the possibility of endocervical adenocarcinoma ( P < .001), leading to preoperative radiation therapy in 4 patients. Median BMI was similar in the LUS and corpus groups. Seventy-three percent of the available LUS tumors had a similar immunohistochemical expression pattern to conventional endometrioid adenocarcinoma. Because of the young median age for the LUS group, we performed immunohistochemistry for Lynch syndrome-associated DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. Microsatellite instability testing (MSI) and MLH1 promoter hypermethylation were performed when indicated. Thirty-six percent of the LUS tumors were MSI-high. Ten of thirty-five (29%) women with LUS tumors were either confirmed to have Lynch Syndrome or were strongly suspected to have Lynch Syndrome based on tissue-based molecular assays (95% CI, 16 to 45%). ^ Conclusions. Endometrial carcinoma arising in the LUS is a clinical and pathologic entity which can be diagnostically confused with cervical adenocarcinoma. In general, LUS tumors can be correctly identified as being endometrial carcinoma using the immunohistochemical panel noted above. The prevalence of Lynch Syndrome in patients with LUS tumors is much greater than that of the general endometrial cancer population (1.8%) or in endometrial cancer patients younger than 50 years of age (8–9%). Based on our results, the possibility of Lynch Syndrome should be considered in women with LUS tumors. ^

Statistical Methods for Differential Expressions of Genes Detected in Multiple-Condition Experiment of Microarray

Most studies of differential gene-expressions have been conducted between two given conditions. The two-condition experimental (TCE) approach is simple in that all genes detected display a common differential expression pattern responsive to a common two-condition difference. Therefore, the genes that are differentially expressed under the other conditions other than the given two conditions are undetectable with the TCE approach. In order to address the problem, we propose a new approach called multiple-condition experiment (MCE) without replication and develop corresponding statistical methods including inference of pairs of conditions for genes, new t-statistics, and a generalized multiple-testing method for any multiple-testing procedure via a control parameter C. We applied these statistical methods to analyze our real MCE data from breast cancer cell lines and found that 85 percent of gene-expression variations were caused by genotypic effects and genotype-ANAX1 overexpression interactions, which agrees well with our expected results. We also applied our methods to the adenoma dataset of Notterman et al. and identified 93 differentially expressed genes that could not be found in TCE. The MCE approach is a conceptual breakthrough in many aspects: (a) many conditions of interests can be conducted simultaneously; (b) study of association between differential expressions of genes and conditions becomes easy; (c) it can provide more precise information for molecular classification and diagnosis of tumors; (d) it can save lot of experimental resources and time for investigators.^

Elevated CO2 alters larval proteome and its phosphorylation status in the commercial oyster, Crassostrea hongkongensis

Ocean acidification (OA) is beginning to have noticeable negative impact on calcification rate, shell structure and physiological energy budgeting of several marine organisms; these alter the growth of many economically important shellfish including oysters. Early life stages of oysters may be particularly vulnerable to OA-driven low pH conditions because their shell is made up of the highly soluble form of calcium carbonate (CaCO3) mineral, aragonite. Our long-term CO2 perturbation experiment showed that larval shell growth rate of the oyster species Crassostrea hongkongensis was significantly reduced at pH < 7.9 compared to the control (8.2). To gain new insights into the underlying mechanisms of low-pH-induced delays in larval growth, we have examined the effect of pH on the protein expression pattern, including protein phosphorylation status at the pediveliger larval stage. Using two-dimensional electrophoresis and mass spectrometry, we demonstrated that the larval proteome was significantly altered by the two low pH treatments (7.9 and 7.6) compared to the control pH (8.2). Generally, the number of expressed proteins and their phosphorylation level decreased with low pH. Proteins involved in larval energy metabolism and calcification appeared to be down-regulated in response to low pH, whereas cell motility and production of cytoskeletal proteins were increased. This study on larval growth coupled with proteome change is the first step toward the search for novel Protein Expression Signatures indicative of low pH, which may help in understanding the mechanisms involved in low pH tolerance.

Analysis of Pacific oyster larval proteome and its response to high-CO2

Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO(2) due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO(2). Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition.

LHPP, the light-harvesting NADPH:protochlorophyllide (Pchlide) oxido¬reductase:Pchlide complex of etiolated plants, is developmentally expressed across the barley leaf gradient

NADPH:protochlorophyllide oxidoreductase is a key enzyme for the light-induced greening of etiolated angiosperm plants. In barley, two POR proteins exist termed PORA and PORB that have previously been proposed to structurally and functionally cooperate in terms of a higher molecular mass light-harvesting complex named LHPP, in the prolamellar body of etioplasts [Nature 397 (1999) 80]. In this study we examined the expression pattern of LHPP during seedling etiolation and de-etiolation under different experimental conditions. Our results show that LHPP is developmentally expressed across the barley leaf gradient. We further provide evidence that LHPP operates both in plants that etiolate completely before being exposed to white light and in plants that etiolate only partially and begin light-harvesting as soon as traces of light become available in the uppermost parts of the soil. As a result of light absorption, in either case LHPP converts Pchlide a to chlorophyllide (Chlide) a and in turn disintegrates. The released Chlide a, as well as Chlide b produced upon LHPP’s light-dependent dissociation, which leads to the activation of the PORA as a Pchlide b-reducing enzyme, then bind to homologs of water-soluble chlorophyll proteins of Brassicaceae. We propose that these proteins transfer Chlide a and Chlide b to the thylakoids, where their esterification with phytol and assembly into the photosynthetic membrane complexes ultimately takes place. Presumably due to the tight coupling of LHPP synthesis and degradation, as well as WSCP formation and photosynthetic membrane assembly, efficient photo-protection is conferred onto the plant.

Nitrate-induced early transcriptional changes during imbibition in non-after-ripened Sisymbrium officinale seeds.

We have here demonstrated for the first time that nitrate not only accelerates testa rupture of non- AR seeds but also modifies expression pattern of the cell-wall remodeling proteins (mannanases; SoMAN6 and SoMAN7) and key genes belonging to metabolism and signaling of ABA (SoNCED6, SoNCED9, SoCYP707A2 and SoABI5) and GAs (SoGA3ox, SoGA20ox, SoGA2ox and SoRGL2). These results were obtained during Sisymbrium officinale seed imbibition in the absence of endosperm rupture. Exogenous ABA induced a notable inhibition of testa rupture in both absence and presence of nitrate being this effect sharply reversed by GA4+7. However, nitrate was capable to provoke testa rupture in absence of ABA synthesis. The expression of SoMAN6 and SoMAN7 were positively altered by nitrate. Although ABA synthesis seems apparent at the start of non-AR seed imbibition, taken together the results of SoNCED6, SoNCED9 and SoCYP707A2 expression seem to suggest that nitrate leads to a strong net ABA decrease. Likewise, nitrate positively affected the SoABI5 expression when the SoNCED9 expression was also stimulated. By contrast, at the early and final of imbibition, nitrate clearly inhibited the SoABI5 expression. The expression of SoGA2ox6 and SoGA3ox2 are strongly inhibited by nitrate whereas of SoGA20ox6 was stimulated. On the other hand, SoRGL2 transcript level decreased in the presence of nitrate. Taken together, the results presented here suggest that the nitrate signaling is already operative during the non-AR S. officinale seeds imbibition. The nitrate, in cross-talk with the AR network likely increases the favorable molecular conditions that trigger germination.

The nitrate-afterripening crosstalk is involved in the testa rupture of sisymbryum offinales seed

The loss of seed dormancy can occur by exposing the seed at low moisture storage conditions (afterripening; AR). Since a positive GA:ABA ratio play a key role in the reactivation of germination of non-dormant seeds, it seems obvious that a remarkable effect of AR is the decreasing of both ABA levels and sensitivity, as well as the increment of GA synthesis and sensitivity. ABA levels are regulated by control both of its biosynthesis thorough the 9-cis-epoxycarotenoid dioxygenase (NCED) encoding genes and its catabolism mediated mainly by ABA-8¿-hydroxylases (CYP707A). On the other hand, the last steps of the GA biosynthesis pathway should be involved to control its levels. Namely, GA20ox and GA3ox catalyzing the biosynthesis of active GA and GA2ox which catalyzes the GA inactivation. The presence of nitrate accelerates the sensu stricto germination of non-AR S. officinale seeds. Here, we demonstrate that in AR seeds nitrate also alters the expression pattern of key genes involved in ABA and GA metabolism and signalling (i.e. SoNCED6, SoNCED9, SoCYP707A2, SoABI5, SoGA3ox2, SoGA20ox6, SoGA2ox6 and SoRGL2). These results suggest that the nitrate signalling is also operative during imbibition of AR S. officinale seeds.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.