Anne Peaston

Systems biology of the 2-cell mouse embryo

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

Epigenetics and phenotypic variation in mammals

What causes phenotypic variation? By now it is clear that phenotype is a result of the interaction between genotype and environment, in addition to variation not readily attributable to either. Epigenetic phenomena associated with phenotypic variation at the biochemical, cellular, tissue, and organism level are now well recognized and are likely to contribute to the “intangible variation” alluded to. While it is clear that epigenetic modifications are mitotically heritable, the fidelity of this process is not well understood. Inheritance through more than one generation of meioses is even less well studied. So it remains unclear to what extent epigenetic changes contribute to phenotypic variation in natural populations. How might such evidence be obtained? What are the features of phenotypes that might suggest an epigenetic component? How much of the epigenetic component is truly independent of genetic changes? The answers to such questions must come from studies designed specifically to detect subtle, stochastically determined phenotypic variation in suitable animal models.

Analysis of gene expression in a developmental context emphasizes distinct biological leitmotifs in human cancers.

BackgroundIn recent years, the molecular underpinnings of the long-observed resemblance between neoplastic and immature tissue have begun to emerge. Genome-wide transcriptional profiling has revealed similar gene expression signatures in several tumor types and early developmental stages of their tissue of origin. However, it remains unclear whether such a relationship is a universal feature of malignancy, whether heterogeneities exist in the developmental component of different tumor types and to which degree the resemblance between cancer and development is a tissue-specific phenomenon.ResultsWe defined a developmental landscape by summarizing the main features of ten developmental time courses and projected gene expression from a variety of human tumor types onto this landscape. This comparison demonstrates a clear imprint of developmental gene expression in a wide range of tumors and with respect to different, even non-cognate developmental backgrounds. Our analysis reveals three classes of cancers with developmentally distinct transcriptional patterns. We characterize the biological processes dominating these classes and validate the class distinction with respect to a new time series of murine embryonic lung development. Finally, we identify a set of genes that are upregulated in most cancers and we show that this signature is active in early development.ConclusionThis systematic and quantitative overview of the relationship between the neoplastic and developmental transcriptome spanning dozens of tissues provides a reliable outline of global trends in cancer gene expression, reveals potentially clinically relevant differences in the gene expression of different cancer types and represents a reference framework for interpretation of smaller-scale functional studies.

Multiple centrosomal microtubule organising centres and increased microtubule stability are early features of VP-16-induced apoptosis in CCRF-CEM cells

Microtubular reorganisation contributing to apoptotic morphology occurs in normal and neoplastic cells undergoing apoptosis induced by cytotoxic drugs [1-3]. The aim of this study was to correlate the changes in the microtubules (MTs) with behavior of the centrosome in apoptotic cells, and to see whether post-translational changes in tubulin occurred with the emergence of apoptotic MT bands. Apoptosis was induced in the human T-cell leukaemia line (CCRF-CEM) by treatment with 17 microM etoposide over a 4 h period. The time course of changes was assessed using flow cytometry (FCM) and immunocytochemistry in cells labelled for a centrosomal antigen (CSP-alpha) or alpha-tubulins. One hour following treatment we observed multiple centrosomal microtubule organising centres (MTOCs) associated with the nucleus and the transient appearance of a subset of stable MTs detected with an antibody specific for acetylated alpha-tubulin, as the bands of MTs which lobulate the nucleus are formed. The altered properties of the MTs thus reflect changes in function as apoptosis progresses.

MRP1 gene expression level regulates the death and differentiation response of neuroblastoma cells.

We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue. In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS). Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression. Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls. Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid. Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death.

Genome plasticity in the mouse oocyte and early embryo

In dissecting the molecules and molecular mechanisms that control mammalian oocyte-to-embryo transition, we found abundant transcripts representing developmentally regulated ERVs (endogenous retroviruses) in mouse oocyte and two-cell stage embryo cDNA libraries. These retrotransposons can act as alternative promoters and first exons for diverse genes, synchronizing their expression. Heritable genetic change due to replication of these retrotransposons probably occurs specifically in oocytes and early embryos. ERVs are usually epigenetically silenced, through DNA methylation and chromatin-based mechanisms. Their activation and silencing indicates a change in the epigenetic state of the genome. The thousands of endogenous retro-elements in the mouse genome provides potential scope for large-scale co-ordinated epigenetic fluctuations and leads to the hypothesis that differential transposable element expression triggers sequential reprogramming of the embryonic genome during the oocyte-to-embryo transition.

Evaluation of Commercially Available Antibodies to Cytokeratin Intermediate Filaments and Laminin in Normal Cat Pinna

The pattern of distribution of cytokeratin (CK) intermediate filaments can be used to characterize subsets of epithelial tissues. The purpose of the study was to examine the CK expression of feline pinna skin. Six normal feline pinnae were routinely processed in formalin. An immunohistochemical method was used to stain the pinnae with 8 commercially available anti-human CK antibodies (Abs) (PKK1, CAM 5.2, UCD 10/ 11, 35BH11, 34BE12, AE1/AE3, MAK 6, A575) and an anti-human laminin Ab. All the CK Abs selectively localized to epithelium except 35BH11, which did not react with any part of the pinna. Some epithelial subsets were identified by their unique staining pattern with CK Abs. Basal cells but not suprabasal cells of the epidermis stained with PKK1; basal but not lumenal cells of apocrine glands stained with 34BE12. Apocrine glands stained with all CK Abs except 35BH11. All epithelial structures were stained with A575. Basal lamina of epithelial and mesenchymal tissues was clearly identified by the anti-laminin Ab. The results indicate that in cat pinna some commercially available anti-human CK Abs selectively stain subsets of epithelium and adnexa. PKK1, 34BE12, and A575 were the CK Abs with the most consistent staining patterns, the other Abs stained more variably from pinna to pinna. The pattern of epithelial and adnexal staining was similar but not identical to that reported for humans.

A Tudor Domain Protein SPINDLIN1 Interacts with the mRNA-Binding Protein SERBP1 and Is Involved in Mouse Oocyte Meiotic Resumption

Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.

1 – Fertilization and Activation of the Embryonic Genome

This chapter discusses the fertilization and activation of the embryonic genome. The full-grown oocyte arrested in prophase of the first meiotic division contains all of the molecules that are utilized to bridge the period of transcriptional silence that begins with the completion of oocyte growth. Under hormonal stimulation the full-grown oocyte begins maturation, completing the first meiosis and the first half of the second meiotic division before arresting in metaphase of the second meiotic division. During this period the extensive stores of maternal messages are selectively utilized, which can result in the synthesis of a new and perhaps a different set of proteins. Simultaneously, preexisting maternal proteins can undergo post-translational modification and degradation. These programmed events result in an oocyte that is ready for fertilization. Fertilization initiates a cascade of events, also dependent on protein modifications and on the timely synthesis of new proteins from maternal mRNA stores that leads to completion of the second meiotic division, remodeling of egg and sperm chromatin, DNA synthesis, entry into the first mitosis, and activation of the embryonic genome.

Adverse Drug Reaction Attributable to Aluminum Phthalocyanine Tetrasulphonate Administration in Domestic Cats

Phthalocyanines are currently under investigation as photosensitizers for use in photodynamic therapy (PDT). We have been examining the efficacy of PDT in Domestic cats that have been treated for nasal and aural squamous cell carcinomas (SCC) using the photosensitizer aluminum phthalocyanine tetrasulphonate (AIPCS,) (Porphyrin Products, Logan, UT). Both clinical treatment and pharmacokinetic experiments have involved a total of 63 random-bred mature cats, considered normal on the basis of physical examinations, pretreatment serum chemistry profiles, and hematologic screens. Cats with positive tests for feline leukemia virus or feline immunodeficiency virus were excluded. Fortyfive of these cats had SCC and were treated one to three times with PDT using AlPCS,. Owners of cats failing therapy were requested to donate their cats for use in pharmacokinetic studies of AlPCS,. Pharmacokinetic studies were also performed using 18 cats that did not have SCC. These cats were obtained solely for pharmacokinetic experiments and received only one AlPCS, injection. All cats in either treatment or pharmacokinetic studies were injected intravenously with 2.0 mg/kg AlPCS,, which was prepared by dissolving AlPCS, in phosphate-buffered saline to a concentration of 2 mg/rnl and adjusting the pH to 7.35 with 0.1 N NaOH. The solution was passed through a 0.45-wm filter prior to injection. There were at least 45 days between injections in cats treated clinically more than once and in cats that failed treatment and entered pharmacokinetic studies. Cats in treatment studies were irradiated 24 hours after injection with 675-nm light from an argon