Genetic assessment of parentage in the caridean rock shrimp Rhynchocinetes typus based on microsatellite markers

Over the past decade, the common rock shrimp, Rhynchocinetes typus H. Milne Edwards, 1837, has been the focus of extensive investigations on mating behaviour. The species is now perceived as a model system for the study of reproductive strategies and sexual conflict in crustaceans displaying external fertilization. Using molecular markers, the current study assesses whether social mating behaviour in common rock shrimp translates into true genetic parentage. In a large mesocosm tank with >200 individuals of both sexes, the analysis of 15 families (22 eggs per female) for three informative microsatellites unambiguously confirmed multiple paternity in 11 instances (73%) involving, in each case, two to four males. Where more than one male was identified siring a particular brood, reproductive skew was apparent towards a single individual. Results suggest that multiple paternity in this species results from subordinate male coercive behaviour, female solicitation of multiple male matings or a combination of both.

The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

Molecular markers for predicting recurrence, progression and outcomes of bladder cancer (do the poster boys need new posters

Purpose of review
Molecular markers for bladder cancer recurrence and
progression continue to drive many research programmes.
Translating the laboratory findings into the clinical environment
where these markers are used in clinical decision making has
proved problematic. In the clinical arena, stage and grade are
still the main focus for decisions about patient management.
There is however an evolution in bladder cancer research from
single-marker/single-pathway research to a more global
assessment of the tumour cell with DNA microarrays and
proteomics.
Recent findings
In the last year, DNA microarray assessment has revealed
several interesting molecular markers such as p33ING1 and
DEK. Parallel ‘conventional’ single-pathway research has
focused on new novel markers such as HER2/neu, survivin and
matrix metalloproteinase 2 (MMP-2). Molecular markers that
have a long-standing association with bladder cancer
progression such as p53, E-cadherin and Ki-67 have been
reviewed by both single-marker studies and by microarray
studies and their status remains important.
Summary
It is an exciting time in the molecular biology research of bladder
cancer as the focus changes to assess the global genetic and
protein expression within tumour cells. From such a wealth of
information it is likely that molecular markers will make the
translation from benchside to bedside.

Heavy metals as markers for assessing environmental pollution from chemical warehouse and plastics fires

Case histories of large, accidental fires are presented to illustrate that heavy metals may be used as markers to assess the extent of localized environmental contamination resulting from fires. Due to the complexity of fire chemistry with respect to organic pollutants, determination of organic pollutants in the environment following a fire would be time consuming and expensive. Using heavy metals as markers on the other hand is much cheaper and can be done very rapidly. © 1995.

The role of secreted frizzled-related protein 2 expression in prostate cancer

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2.

METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients.

CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.

Epidermolysis bullosa:evidence for linkage to genetic markers on chromosome 1 in a family with the autosomal dominant simplex form

DNA from members of a three-generation pedigree of Irish origin, displaying an autosomal dominant simplex form of epidermolysis bullosa of the epidermolytic, simplex, or Koebner variety (EBS2), was analyzed for linkage with a set of markers derived from the long arm of chromosome 1. Two-point analysis revealed positive lod scores for five of these markers, AT3 (Z = 2.107, theta = 0), APOA2 (Z = 1.939, theta = 0.15), D1S66 (Z = 1.204, theta = 0), D1S13 (Z = 1.026, theta = 0.15), and D1S65 (Z = 0.329, theta = 0.15). Multilocus analysis, incorporating the markers D1S19, D1S16, D1S13, APOA2, D1S66, AT3, and D1S65, resulted in a lod score of 3 maximizing at AT3. These data strongly support previous tentative indications of linkage between EBS2 and genetic markers on the long arm of chromosome 1.

Major and trace elements in milk and Halloumi cheese as markers for authentication of goat feeding regimes and geographical origin

Sixty samples of milk, Halloumi cheese and local grazing plants (i.e. shrubs) were collected over a year from dairy farms located on three different locations of Cyprus. Major and trace elements were quantified using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Milk and Halloumi cheese produced in different geographical locations presented significant differences in the concentration of some of the elements analysed. Principal component analysis showed grouping of samples according to the region of production for both milk and cheese samples. These findings show that the assay of elements can provide useful fingerprints for the characterisation of dairy products.

O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

Terminal and progenitor lineage-survival oncogenes as cancer markers

Tumour classification has traditionally focused on differentiation and cellular morphology, and latterly on the application of genomic approaches. By combining chromatin immunoprecipitation with expression array, it has been possible to identify direct gene targets for transcription factors for nuclear hormone receptors. At the same time, there have been great strides in deriving stem and progenitor cells from tissues. It is therefore timely to propose that pairing the isolation of these cell subpopulations from tissues and tumours with these genomics approaches will reveal conserved gene targets for transcription factors. By focusing on transcription factors (lineage-survival oncogenes) with roles in both organogenesis and tumourigenesis at multiple organ sites, we suggest that this comparative genomics approach will enable developmental biology to be used more fully in relation to understanding tumour progression and will reveal new cancer markers. We focus here on neurogenesis and neuroendocrine differentiation in tumours.

Pro-neural transcription factors as cancer markers

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours.

RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers.

CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.