Ritu Pandey

Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil.

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.

Comparative Analysis of SET Domain Proteins in Maize and Arabidopsis Reveals Multiple Duplications Preceding the Divergence of Monocots and Dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

Pathway Miner: extracting gene association networks from molecular pathways for predicting the biological significance of gene expression microarray data

UNLABELLED We have developed a web-based system (Pathway Miner) for visualizing gene expression profiles in the context of biological pathways. Pathway Miner catalogs genes based on their role in metabolic, cellular and regulatory pathways. A Fisher exact test is provided as an option to rank pathways. The genes are mapped onto pathways and gene product association networks are extracted for genes that co-occur in pathways. The networks can be filtered for analysis based on user-selected options. AVAILABILITY Pathway Miner is a freely available web accessible tool at http://www.biorag.org/pathway.html

Loss of primary cilia occurs early in breast cancer development

BackgroundPrimary cilia are microtubule-based organelles that protrude from the cell surface. Primary cilia play a critical role in development and disease through regulation of signaling pathways including the Hedgehog pathway. Recent mouse models have also linked ciliary dysfunction to cancer. However, little is known about the role of primary cilia in breast cancer development. Primary cilia expression was characterized in cancer cells as well as their surrounding stromal cells from 86 breast cancer patients by counting cilia and measuring cilia length. In addition, we examined cilia expression in normal epithelial and stromal cells from reduction mammoplasties as well as histologically normal adjacent tissue for comparison.ResultsWe observed a statistically significant decrease in the percentage of ciliated cells on both premalignant lesions as well as in invasive cancers. This loss of cilia does not correlate with increased proliferative index (Ki67-positive cells). However, we did detect rare ciliated cancer cells present in patients with invasive breast cancer and found that these express a marker of basaloid cancers that is associated with poor prognosis (Cytokeratin 5). Interestingly, the percentage of ciliated stromal cells associated with both premalignant and invasive cancers decreased when compared to stromal cells associated with normal tissue. To understand how cilia may be lost during cancer development we analyzed the expression of genes required for ciliogenesis and/or ciliary function and compared their expression in normal versus breast cancer samples. We found that expression of ciliary genes were frequently downregulated in human breast cancers.ConclusionsThese data suggest that primary cilia are lost early in breast cancer development on both the cancer cells and their surrounding stromal cells.

The effects of endocrine and mechanical stimulation on stage I lactogenesis in bovine mammary epithelial cells

The study objective was to evaluate the effect of endocrine and mechanical (gel release) signaling on bovine mammary epithelial cell ultrastructure and gene expression. Cultures receiving only one stimulus demonstrated partially differentiated ultrastructure, which included abundant polysomes, limited rough endoplasmic reticulum, and absence of secretory products, whereas the 2 stimuli together induced a more complete lactogenic phenotype that included increased rough endoplasmic reticulum, abundant lipid droplets, and secretory vesicles containing casein micelles. The structural data indicated that although synthesis of milk components was initiated, the copious synthesis and secretion associated with stage II lactogenesis was not evident. Microarray analysis revealed that both prolactin and gel release independently regulated several genes linked to a wide array of cellular activities. In combination, they regulated fewer genes targeted to lactogenesis. Genes regulated by the combination treatment included claudin 7, multiple caseins, xanthine oxidoreductase, and several protein synthesis, packaging, and transport genes. Genes related to structural activity including keratin 15 (morphogenesis), alpha-spectrin (cell shape via actin cytoskeleton), and chitinase-like protein 1 (tissue remodeling) were up-regulated by the combination treatment as was the transcription factor Kruppel-like factor 2 (KLF-2). However, Snail 2, which down-regulates and inhibits tight junction components, was repressed in response to the combination treatment. These results suggest coordination between endocrine and physical signals at the genomic level that produces a more specific and targeted transcriptional response associated with stage I lactogenesis. A molecular pathway analysis of the differentially expressed genes revealed that genes regulating cell signaling were linked to those regulating cell structure and adhesion.

Using bioinformatics and genome analysis for new therapeutic interventions

The genome era provides two sources of knowledge to investigators whose goal is to discover new cancer therapies: first, information on the 20,000 to 40,000 genes that comprise the human genome, the proteins they encode, and the variation in these genes and proteins in human populations that place individuals at risk or that occur in disease; second, genome-wide analysis of cancer cells and tissues leads to the identification of new drug targets and the design of new therapeutic interventions. Using genome resources requires the storage and analysis of large amounts of diverse information on genetic variation, gene and protein functions, and interactions in regulatory processes and biochemical pathways. Cancer bioinformatics deals with organizing and analyzing the data so that important trends and patterns can be identified. Specific gene and protein targets on which cancer cells depend can be identified. Therapeutic agents directed against these targets can then be developed and evaluated. Finally, molecular and genetic variation within a population may become the basis of individualized treatment.

Ultraviolet-B Induced Changes in Ultrastructure and D1/D2 Proteins in Cyanobacteria Synechococcus sp. PCC 7942

Effects of ultraviolet-B (UV-B) irradiation on ultrastructure, total cellular protein, and PS2 proteins D1 and D2 of Synechococcus sp. PCC 7942 cells was studied. The scanning electron micrographs showed UV-B radiation induced bending of the cells. The transmission electron micrographs revealed disorganization and shift in thylakoid lamellar structure to one side of the cell. The cellular phycocyanin/chlorophyll ratio decreased with increasing UV-B treatment and due to this the colour of cells turned light-green. No apparent change in total cellular proteins was evident, but the contents of two major proteins of PS2, D1 and D2, showed decline due to UV-B irradiation, although to different extent.

Specific microRNA–mRNA Regulatory Network of Colon Cancer Invasion Mediated by Tissue Kallikrein–Related Peptidase 6

Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P = .005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer. Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

Pilot study on the effects of dietary conjugated linoleic acid on tumorigenesis and gene expression in PyMT transgenic mice

Conjugated linoleic acid (CLA) is a class of commercially available fatty acids that have been associated with anticancer properties in rodent models of chemical carcinogenesis. We conducted a pilot study to examine the antitumor effect of dietary CLA in a polyoma virus-middle T antigen (PyMT) mouse model of invasive breast cancer. Virgin 4-week-old PyMT mice were administered a mixed-isomer CLA diet (1% wt/wt) or control AIN-93G diet for 4 weeks (N = 6 and 5, respectively) and tumor burden was assessed at 8 weeks of age. Thoracic mammary glands were prepared as whole mounts with other glands being formalin fixed and paraffin embedded for histology and immunohistochemistry (IHC). Total RNA was prepared for microarray and real-time reverse transcription-polymerase chain reaction analysis. Western blots were performed for protein expression analysis. Tumor incidence was significantly increased in CLA-treated animals compared with controls (P = 0.009) and occurred with extensive lobular-alveolar expansion and loss of mammary adipose tissue. More than 100 genes were downregulated > or = 2-fold in the CLA-treated group compared with controls, including adipose-specific markers, as wells as cytoskeletal and adhesion-related genes. This was supported by dramatic decreases in the epithelial adherens E-cadherin and beta-catenin as demonstrated by IHC. Taken together, these results suggest that dietary CLA affects the mammary stromal environment, leading to tumor progression and cellular expansion in the PyMT mouse model. Further studies of the potential for cancer promotion are needed, especially because mixed-isomer CLA formulations are sold commercially as a nutritional supplement.

Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.