Acquired resistance of pancreatic cancer cells to cisplatin is multifactorial with cell context-dependent involvement of resistance genes.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of malignancies, in large measure, due to the propensity of PDAC cells to acquire resistance to chemotherapeutic agents. A better understanding of the molecular basis of acquired resistance is a major focus of contemporary PDAC research. We report here the results of a study to independently develop cisplatin resistance in two distinct parental PDAC cell lines, AsPC1 and BxPC3, and to subsequently examine the molecular mechanisms associated with the acquired resistance. Cisplatin resistance in both resistant cell lines was found to be multifactorial and to be associated with mechanisms related to drug transport, drug inactivation, DNA damage response, DNA repair and the modulation of apoptosis. Our results demonstrate that the two resistant cell lines employed alternative molecular strategies in acquiring resistance dictated, in part, by pre-existing molecular differences between the parental cell lines. Collectively, our findings indicate that strategies to inhibit or reverse acquired resistance of PDAC cells to cisplatin, and perhaps other chemotherapeutic agents, may not be generalized but will require individual molecular profiling and analysis to be effective.

Evidence for the role of microRNA 374b in acquired cisplatin resistance in pancreatic cancer cells.

Recent evidence has implicated microRNAs (miRNAs) as potentially significant players in the acquisition of cancer-drug resistance in pancreatic and other cancers. To evaluate the potential contribution of miRNAs in acquired resistance to cisplatin in pancreatic cancer, we compared levels of more than 2000 human miRNAs in a cisplatin-resistant cell line (BxPC3-R) derived from parental (BxPC3) cells by step-wise exposure to increasing concentrations of the drug over more than 20 passages. The acquired drug resistance was accompanied by significant changes in the expression of 57 miRNAs, of which 23 were downregulated and 34 were upregulated. Employing a hidden Markov model (HMM) algorithm, we identified downregulation of miR-374b as likely being directly involved in acquisition of the drug-resistant phenotype. Consistent with this prediction, ectopic overexpression of miR-374b in the resistant BxPC3-R cells restored cisplatin sensitivity to levels approaching those displayed by the BxPC3 parental cells. The results are consistent with a growing body of evidence implicating miRNAs in acquired cancer-drug resistance and with the potential therapeutic value of these small regulatory RNAs in blocking and/or reversing the process.

Clinical selection criteria can predict futile intervention in patients referred for percutaneous endoscopic gastrostomy insertion.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) placement is performed in a patient group with high mortality in the short and medium term. For a significant proportion of patients, the procedure provides no increase in survival. There are no standardised assessment tools available to determine the clinical appropriateness of PEG placement, nor any to predict clinical outcome. AIM: The study aims to determine whether clinical assessment, by a trained dietitian, of the appropriateness of PEG placement is predictive of mortality in the short and medium terms. METHODS: A prospective audit was undertaken of all requests for PEG placement at a single large, publicly funded Australian tertiary hospital. The clinical appropriateness of each request was assessed by a trained dietitian, and data on age, sex, reason for referral, comorbidities and satisfaction of assessment criteria were collected, and patient outcome and survival were compared for all patients according to whether a PEG was inserted or not. Main outcome measures were mortality at 30 and 150 days after referral. RESULTS: During the period 2005-2008, 198 patients were referred for PEG; 94 were assessed as appropriate referrals, 104 as inappropriate. Eighty-four patients who underwent gastrostomy, after being assessed as appropriate, had significantly reduced mortality at 30 days (96.4% vs 74.6%, P < 0.0001) and 150 days (82.1% vs 57.9%, P = 0.0001) compared with all other patients. Patients who received PEG despite contrary advice had no significant survival advantage, at 30 days or 150 days, over patients who did not receive PEG. CONCLUSION: The application of selection criteria by trained assessors improves patient selection for PEG insertion and predicts mortality at early and later time points, by identifying patients unlikely to benefit from PEG. The group of patients who received a gastrostomy despite an adverse assessment had no mortality benefit - in these patients, the procedure may have been futile.

Design of high efficiency multi-GHz SiGe HBT electro-optic modulator.

We design and theoretically analyze a heterojunction bipolar transistor (HBT) electro-optic (EO) modulator with a composition graded SiGe base. The waveguide has a large cross-section of 1 microm for ease of fiber alignment. At a base-emitter bias of V BE = 2.5 V, a pi-phase shift requires 74.5 microm interaction length for TM polarization at lambda = 1.55 microm. The total optical attenuation is 3.9 dB to achieve a pi-phase shift in this condition. This device is expected to operate at a switching speed of 2.4 GHz.

The design, synthesis and anticancer activity of new nitrogen mustard derivatives of natural indole phytoalexin 1-methoxyspirobrassinol.

Nitrogen mustards cis-1-methoxy-2-deoxy-2-[N,N-bis(2 -chloroethyl)amino]spirobrassinol (4) and trans-1-methoxy-2-deoxy-2-[N,N-bis(2 -chloroethyl)amino]spirobrassinol (5) derived from 1-methoxyspirobrassinol, an indole phytoalexin produced by the Japanese radish Raphanus sativus var. hortensis were designed as prospective dual-action compounds with DNA-alkylating effect and glutathione-depleting effects that may sensitize cancer cells to alkylating agents. Both new compounds demonstrated cytostatic/cytotoxic effects on various leukemia and ovarian cancer cell lines and dsDNA-destabilizing effects in vitro. Compound 4, the more promising of the two compounds, exerts earlier onset of anticancer effects on Jurkat cells via induction of apoptosis compared to the traditional alkylating anticancer agent melphalan. In addition, it demonstrated higher potency on ovarian cancer OVCAR-3 cell line and lower fold resistance between Jurkat and Jurkat-M cells selected for the resistance to melphalan. Therefore, compound 4 may be less affected by certain cancer drug resistance mechanisms than melphalan and it may become a prototype of a new class of anticancer active nitrogen mustards that combine DNA-damaging and DNA-damage-sensitizing properties.

Host defenses to transposable elements and the evolution of genomic imprinting.

Genomic imprinting is the differential expression of maternally and paternally inherited alleles of specific genes. Several organismic level hypotheses have been offered to explain the evolution of genomic imprinting. We argue that evolutionary explanations of the origin of imprinting that focus exclusively on the organismic level are incomplete. We propose that the complex molecular mechanisms that underlie genomic imprinting originally evolved as an adaptive response to the mutagenic potential of transposable elements (TEs). We also present a model of how these mechanisms may have been co-opted by natural selection to evolve molecular features characteristic of genomic imprinting.

An amyloid-like C-terminal domain of thrombospondin-1 displays CD47 agonist activity requiring both VVM motifs.

Two VVM-containing peptides in the C-terminal domain (CBD) of thrombospondin-1 function as CD47 agonists. A recombinant form of the CBD (rCBD) has been expressed that contains both VVM sites and exhibits CD47-dependent binding of C32 melanoma cells when coated at concentrations 100x lower than the peptide 4N1K (kRFYVVMWKk). Circular dichroism and thioflavin T binding of a recombinant form of the C-terminal domain (rCBD) of thrombospondin-1 indicated a species highly enriched in beta-sheet secondary structure, with spectra similar to those of amyloid proteins. Reduction of the CD signal with progressively higher concentrations of guanidine hydrochloride was correlated with a loss of cell-binding activity. Melanoma cell spreading on vitronectin was strongly stimulated by immobilized rCBD co-coated at concentrations more than 50x lower than 4N1K, and the effect was blocked by treatment with pertussis toxin, consistent with the known mediation of CD47 signaling by trimeric G(i). Mutations of either or both VV sequences of rCBD (1037-38 and 1123-24 of TSP1) to GG had a modest effect on cell binding, a component of which was inhibited by heparin. However, all three mutants dramatically reduced the signaling-dependent stimulation of cell spreading, indicating that the VVM motifs of rCBD are structurally linked in CD47 activation.

Evolutionary history of Cer elements and their impact on the C. elegans genome.

We report the results of sequence analysis and chromosomal distribution of all distinguishable long terminal repeat (LTR) retrotransposons (Cer elements) in the Caenorhabditis elegans genome. Included in this analysis are all readily recognizable full-length and fragmented elements, as well as solo LTRs. Our results indicate that there are 19 families of Cer elements, some of which display significant subfamily structure. Cer elements can be clustered based on their tRNA primer binding sites (PBSs). These clusters are in concordance with our reverse transcriptase- and LTR-based phylogenies. Although we find that most Cer elements are located in the gene depauperate chromosome ends, some elements are located in or near putative genes and may contribute to gene structure and function. The results of RT-PCR analyses are consistent with this prediction.

Drosophila euchromatic LTR retrotransposons are much younger than the host species in which they reside.

The recent release of the complete euchromatic genome sequence of Drosophila melanogaster offers a unique opportunity to explore the evolutionary history of transposable elements (TEs) within the genome of a higher eukaryote. In this report, we describe the annotation and phylogenetic comparison of 178 full-length long terminal repeat (LTR) retrotransposons from the sequenced component of the D. melanogaster genome. We report the characterization of 17 LTR retrotransposon families described previously and five newly discovered element families. Phylogenetically, these families can be divided into three distinct lineages that consist of members from the canonical Copia and Gypsy groups as well as a newly discovered third group containing BEL, mazi, and roo elements. Each family consists of members with average pairwise identities > or =99% at the nucleotide level, indicating they may be the products of recent transposition events. Consistent with the recent transposition hypothesis, we found that 70% (125/178) of the elements (across all families) have identical intra-element LTRs. Using the synonymous substitution rate that has been calculated previously for Drosophila (.016 substitutions per site per million years) and the intra-element LTR divergence calculated here, the average age of the remaining 30% (53/178) of the elements was found to be 137,000 +/-89,000 yr. Collectively, these results indicate that many full-length LTR retrotransposons present in the D. melanogaster genome have transposed well after this species diverged from its closest relative Drosophila simulans, 2.3 +/-.3 million years ago.

Genomic analysis of Caenorhabditis elegans reveals ancient families of retroviral-like elements.

Retrotransposons are the most abundant and widespread class of eukaryotic transposable elements. The recent genome sequencing of Caenorhabditis elegans has provided an unprecedented opportunity to analyze the evolutionary relationships among the entire complement of retrotransposons within a multicellular eukaryotic organism. In this article we report the results of an analysis of retroviral-like long terminal repeat retrotransposons in C. elegans that indicate that this class of elements may be even more abundant and divergent than previously expected. The unexpected presence, in C. elegans, of an element displaying a number of characteristics previously thought to be unique to vertebrate retroviruses suggests an ancient lineage for this important class of infectious agents.

Evidence for the recent horizontal transfer of long terminal repeat retrotransposon.

The evolutionary dynamics existing between transposable elements (TEs) and their host genomes have been likened to an "arms race." The selfish drive of TEs to replicate, in turn, elicits the evolution of host-mediated regulatory mechanisms aimed at repressing transpositional activity. It has been postulated that horizontal (cross-species) transfer may be one effective strategy by which TEs and other selfish genes can escape host-mediated silencing mechanisms over evolutionary time; however, to date, the most definitive evidence that TEs horizontally transfer between species has been limited to class II or DNA-type elements. Evidence that the more numerous and widely distributed retroelements may also be horizontally transferred between species has been more ambiguous. In this paper, we report definitive evidence for a recent horizontal transfer of the copia long terminal repeat retrotransposon between Drosophila melanogaster and Drosophila willistoni.

Genomic demography: a life-history analysis of transposable element evolution.

Retrotransposons are ubiquitous mobile genetic elements that have played a significant role in shaping eukaryotic genome evolution. The genome of the yeast Saccharomyces cerevisiae harbours five families of retrotransposons, Ty1-Ty5. With the publication of the S. cerevisiae genome sequence, for the first time a full genomic complement of retrotransposon sequences is available. Analysis of these sequences promises to yield insight into the nature of host--transposon coevolution. Evolutionary change in Ty elements depends on their replication and excision rates, which have been determined in the laboratory. Rates measured in the laboratory may differ from those that have operated over evolutionary time. Based on an analysis of sequence data for the Ty1, Ty2 and hybrid Ty1/2 families, we develop a novel 'genomic demography' model to estimate long-term transposition and excision rates and to estimate how long ago these elements entered the yeast genome. We find that rates of excision and transposition have averaged 7.2-8.7 x 10(-8) per generation over evolutionary time. Two separate models provide upper- and lower-bound estimates for the age of the system, suggesting that the first elements entered the genome between approximately 50 million and 250 million generations ago.

The role of interelement selection in Saccharomyces cerevisiae Ty element evolution.

Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes. The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside. Elements with higher rates of genomic replication possess a selective advantage over less active elements. Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements. To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed. The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data. In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons. The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies. These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages. We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside.

Tempo and mode of Ty element evolution in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains five families of long terminal repeat (LTR) retrotransposons, Ty1-Ty5. The sequencing of the S. cerevisiae genome provides an unprecedented opportunity to examine the patterns of molecular variation existing among the entire genomic complement of Ty retrotransposons. We report the results of an analysis of the nucleotide and amino acid sequence variation within and between the five Ty element families of the S. cerevisiae genome. Our results indicate that individual Ty element families tend to be highly homogenous in both sequence and size variation. Comparisons of within-element 5' and 3' LTR sequences indicate that the vast majority of Ty elements have recently transposed. Furthermore, intrafamily Ty sequence comparisons reveal the action of negative selection on Ty element coding sequences. These results taken together suggest that there is a high level of genomic turnover of S. cerevisiae Ty elements, which is presumably in response to selective pressure to escape host-mediated repression and elimination mechanisms.

Comparative genomics and evolutionary dynamics of Saccharomyces cerevisiae Ty elements.

The availability of the complete genome sequence of Saccharomyces cerevisiae provides the unique opportunity to study an entire genomic complement of retrotransposons from an evolutionary perspective. There are five families of yeast retrotransposons, Ty1-Ty5. We have conducted a series of comparative sequence analyses within and among S. cerevisiae Ty families in an effort to document the evolutionary forces that have shaped element variation. Our results indicate that within families Ty elements vary little in terms of both size and sequence. Furthermore, intra-element 5'-3' long terminal repeat (LTR) sequence comparisons indicate that almost all Ty elements in the genome have recently transposed. For each family, solo LTR sequences generated by intra-element recombination far outnumber full length insertions. Taken together, these results suggest a rapid genomic turnover of S. cerevisiae Ty elements. The closely related Ty1 and Ty2 are the most numerous elements in the genome. Phylogenetic analysis of full length insertions reveals that reverse transcriptase mediated recombination between Ty1 and Ty2 elements has generated a number of hybrid Ty1/2 elements. These hybrid Ty1/2 elements have similar genomic structures with chimeric LTRs and chimeric TYB (pol) genes. Analysis of the levels of nonsynonymous (Ka) and synonymous (Ks) nucleotide variation indicates that Ty1 and Ty2 coding regions have been subject to strong negative (purifying) selection. Distribution of Ka and Ks on Ty1, Ty2 and Ty1/2 phylogenies reveals evidence of negative selection on both internal and external branches. This pattern of variation suggests that the majority of full length Ty1, Ty2 and Ty1/2 insertions represent active or recently active element lineages and is consistent with a high level of genomic turnover. The evolutionary dynamics of S. cerevisae Ty elements uncovered by our analyses are discussed with respect to selection among elements and the interaction between the elements and their host genome.

Molecular domestication--more than a sporadic episode in evolution.

Transposable elements are short but complex pieces of DNA or RNA containing a streamlined minimal-genome with the capacity for its selfish replication in a foreign genomic environment. Cis-regulatory sections within the elements orchestrate tempo and mode of TE expression. Proteins encoded by TEs mainly direct their own propagation within the genome by recruitment of host-encoded factors. On the other hand, TE-encoded proteins harbor a very attractive repertoire of functional abilities for a cell. These proteins mediate excision, replication and integration of defined DNA fragments. Furthermore, some of these proteins are able to manipulate important host factors by altering their original function. Thus, if the host genome succeeds in domesticating such TE-encoded proteins by taming their 'anarchistic behavior,' such an event can be considered as an important evolutionary innovation for its own benefit. In fact, the domestication of TE-derived cis-regulatory modules and protein coding sections took place repeatedly in the course of genome evolution. We will present prominent cases that impressively demonstrate the beneficial impact of TEs on host biology over evolutionary time. Furthermore, we will propose that molecular domestication might be considered as a resumption of the same evolutionary process that drove the transition from 'primitive genomes' to 'modern' ones at the early dawn of life, that is, the adaptive integration of a short piece of autonomous DNA into a complex regulatory network.

Interelement selection in the regulatory region of the copia retrotransposon.

We report the results of an analysis of naturally occurring cis-regulatory variation within and between two families of the copia Drosophila long terminal repeat (LTR) retrotransposon. The copia 5' LTR and adjacent untranslated leader region (ULR) consists of a number of well-characterized sequence motifs which play a role in regulating expression of the element. In order to understand the evolutionary forces which may be responsible for generating and maintaining copia regulatory sequence variation, we have quantified levels of naturally occurring copia LTR-ULR nucleotide variation and subjected the data to a series of tests of neutrality. Our analysis indicates that the copia LTR-ULR has been subject to negative purifying selection within families and positive adaptive selection between families. We discuss these findings with respect to the regulatory evolution of retrotransposons and the phenomenon of interelement selection.

Evolution of the copia retrotransposon in the Drosophila melanogaster species subgroup.

We report the results of a phylogenetic survey of the retrotransposon copia in the melanogaster subgroup of the Drosophila genus. The polymerase chain reaction was used to amplify the copia 5' long terminal repeat and the adjacent untranslated leader region from representative melanogaster subgroup species. Restriction and sequence analyses of this region reveal discrete classes of copia size variants within the melanogaster subgroup. Phylogenetic comparisons of copia sequence data indicate that the size variants represent different copia subfamilies which diverged prior to their distribution in the melanogaster subgroup. Our results also suggest that copia elements have been subject to horizontal and vertical transmission during their evolution.

Evidence for the role of recombination in the regulatory evolution of Saccharomyces cerevisiae Ty elements.

The recent completion of the sequencing of the Saccharomyces cerevisiae genome provides a unique opportunity to analyze the evolutionary relationships existing among the entire complement of retrotransposons residing within a single genome. In this article we report the results of such an analysis of two closely related families of yeast long terminal repeat (LTR) retrotransposons, Ty1 and Ty2. In our study, we analyzed the molecular variation existing among the 32 Ty1 and 13 Ty2 elements present within the S. cerevisiae genome recently sequenced within the context of the yeast genome project. Our results indicate that while the Ty1 family is most likely ancestral to Ty2 elements, both families of elements are relatively recent components of the S. cerevisiae genome. Our results also indicate that both families of elements have been subject to purifying selection within their protein coding regions. Finally, and perhaps most interestingly, our results indicate that a relatively recent recombination event has occurred between Ty2 and a subclass of Ty1 elements involving the LTR regulatory region. We discuss the possible biological significance of these findings and, in particular, how they contribute to a better overall understanding of LTR retrotransposon evolution.