Isolation and characterization of the retinoblastoma protein from fish

The retinoblastoma (Rb) gene represents the first tumor suppressor gene characterized. The encoded protein, pRb, plays a crucial role in cell cycle control, preventing malignant cell proliferation. Recently, homologues of the Rb gene have been isolated in fish and the pocket domain, which is central to Rb function, was conserved. In our studies, using coelocanth (Latimeria chalumnae), rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes) and English sole (Parophrys vetulus), we have developed a simple protocol for the isolation of the Rb tumor suppressor protein and determined its' tissue and cellular localization. Fish Rb proteins display apparent molecular weights in the range of 100-110 kDa, similar to the human pRb. The protein was detected in all tissues examined, consistent with the proteins' universal role in cellular signalling. An interesting pattern of immunoreactive bands was detected in each of the cells' two main compartments, suggesting differential proteolysis. Immuno-analysis of the pRb in trout liver tumor material revealed an additional Rb reactive product that was absent in normal liver cell extracts.     

Hepatic Tumors in Flatfish from Sivuch'ya Bight (Peter the Great Bay, Sea of Japan)

The livers of six flatfish species from Sivuch'ya Bight were examined histologically for the presence of neoplastic lesions. Out of 120 fish studied, hepatic tumors were found in two individuals: the black plaice Pleuronectes obscurusand the longsnout flounderPleuronectes punctatissimus.Based on presence of tissue and cellular atypism, tumor pearls surrounded by squamous cells, and absence of mucous secretion, the neoplasm in P. punctatissimuswas classified as squamous cell carcinoma. Abnormal growth of fibrous stroma suggests a scirrhous variant of carcinoma. Tumor in P. obscuruswas classified as a hepatocellular adenoma. This is the first information on liver tumors in flatfishes of Sivuch'ya Bight.     

Tumor suppressor genes

The retinoblastoma sensitivity protein (Rb) and the p53 gene product both appear to function as negative regulators of cell division or abnormal cellular growth in some differentiated cell types. Several types of cancers have been shown to be derived from cells that have extensively mutated both alleles of one or both of these genes, resulting in a loss-of-function mutation. In the case of the p53 gene, this mutational process appears to occur in two steps, with the first mutation at the p53 locus resulting in a trans-dominant phenotype. The mutant p53 gene product enters into an oligomeric protein complex with the wild-type p53 protein derived from the other normal allele and such a complex is inactive or less efficient in its negative regulation of growth control. This intermediate stage of carcinogenesis selects for the proliferation of cells with one mutant allele, enhancing the probability of obtaining a cancer cell with both alleles damaged. The DNA tumor viruses have evolved mechanisms to interact with the Rb and p53 negative regulators of cellular growth in order to enhance their own replication in growing cells. SV40 and adenovirus type 5 produce viral encoded proteins that also form oligomeric protein complexes with p53 and Rb, presumably inactivating their functions. These viral proteins are also the oncogene products of these viruses. Thus, the mechanisms by which cancer may arise in a host, via mutations or virus infections, have fundamental common pathways effecting the same cellular genes and gene products; Rb and p53.     

Long-term dynamics of species composition and abundance of flatfish in waters off Primorye (the Sea of Japan)

Based on the data of trawl surveys, changes in the structure of species composition and flatfish abundance (Pleuronectidae) in waters off Primorye are shown in the period embracing over 20 years (1983 to 2004). In waters off Primorye, 14 flatfish species were recorded in catches. At the present time, the structure of the flatfish part of the fish community in waters off Primorye has undergone considerable changes in comparison with the 1980s: the proportion of deepwater flatfish has considerably increased against the background of a decrease in the abundance of coastal species. A general decrease in the numbers and biomass of flatfish in waters off Primorye is recorded, which is caused by natural reasons against the background of an inconsiderable fishery intensity.     

Feeding biology of the flatfish Citharichthys spilopterus (Bothidae) in a tropical estuary of Mexico

The flatfish Citharichthys spilopterus Günther 1862 is the most common bothid in the coastal lagoons of the Gulf of Mexico. The objective of the present study was to describe the trophic biology of this species in the tropical coastal lagoon of Tampamachoco, Mexico. For the diet analysis, we used multivariate discriminant analysis and trophic niche breadth. The morphological analysis showed that the features of the bucco-pharyngeal cavity, the large stomach and the short intestine of this flatfish relate to a carnivorous habit. The overall diet showed that this species is a third-order consumer, feeding mainly on fish (52%, mainly gobies) and crustaceans (36%, mostly decapods). Diet and trophic niche breadth showed no significant differences between sexes (P > 0.1). By contrast, there were significant ontogenetic differences in the diet and trophic niche breadth, where larger C. spilopterus were almost entirely piscivorous and showed the narrowest trophic niche. As the flatfish grew in size there was a trend toward the consumption of larger prey. The importance of copepods and peracarids correlated inversely with flatfish size (P < 0.001), but fish prey correlated directly to flatfish size (P < 0.001). Likewise, there was an inverse significant correlation between niche trophic breadth and flatfish size (P < 0.005). There were also significant seasonal differences in the diet (P < 0.02), related to the availability and vulnerability of prey in the lagoon during the dry and rainy seasons. However, the trophic niche showed no significant differences between seasons. Finally, we discuss the advantages of discriminant analysis applied to evaluate differences among diets of fish groups when compared with other bivariate and multivariate techniques.     

Expression changes in the stroma of prostate cancer predict subsequent relapse

Biomarkers are needed to address overtreatment that occurs for the majority of prostate cancer patients that would not die of the disease but receive radical treatment. A possible barrier to biomarker discovery may be the polyclonal/multifocal nature of prostate tumors as well as cell-type heterogeneity between patient samples. Tumor-adjacent stroma (tumor microenvironment) is less affected by genetic alteration and might therefore yield more consistent biomarkers in response to tumor aggressiveness. To this end we compared Affymetrix gene expression profiles in stroma near tumor and identified a set of 115 probe sets for which the expression levels were significantly correlated with time-to-relapse. We also compared patients that chemically relapsed shortly after prostatectomy (<1 year), and patients that did not relapse in the first four years after prostatectomy. We identified 131 differentially expressed microarray probe sets between these two categories. 19 probe sets (15 genes overlapped between the two gene lists with p<0.0001). We developed a PAM-based classifier by training on samples containing stroma near tumor: 9 rapid relapse patient samples and 9 indolent patient samples. We then tested the classifier on 47 different samples, containing 90% or more stroma. The classifier predicted the risk status of patients with an average accuracy of 87%. This is the first general tumor microenvironment-based prognostic classifier. These results indicate that the prostate cancer microenvironment exhibits reproducible changes useful for predicting outcomes for patients.     

Is cross-validation better than resubstitution for ranking genes?

MOTIVATION: Ranking gene feature sets is a key issue for both phenotype classification, for instance, tumor classification in a DNA microarray experiment, and prediction in the context of genetic regulatory networks. Two broad methods are available to estimate the error (misclassification rate) of a classifier. Resubstitution fits a single classifier to the data, and applies this classifier in turn to each data observation. Cross-validation (in leave-one-out form) removes each observation in turn, constructs the classifier, and then computes whether this leave-one-out classifier correctly classifies the deleted observation. Resubstitution typically underestimates classifier error, severely so in many cases. Cross-validation has the advantage of producing an effectively unbiased error estimate, but the estimate is highly variable. In many applications it is not the misclassification rate per se that is of interest, but rather the construction of gene sets that have the potential to classify or predict. Hence, one needs to rank feature sets based on their performance. RESULTS: A model-based approach is used to compare the ranking performances of resubstitution and cross-validation for classification based on real-valued feature sets and for prediction in the context of probabilistic Boolean networks (PBNs). For classification, a Gaussian model is considered, along with classification via linear discriminant analysis and the 3-nearest-neighbor classification rule. Prediction is examined in the steady-distribution of a PBN. Three metrics are proposed to compare feature-set ranking based on error estimation with ranking based on the true error, which is known owing to the model-based approach. In all cases, resubstitution is competitive with cross-validation relative to ranking accuracy. This is in addition to the enormous savings in computation time afforded by resubstitution.     

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.     

Convergent signs of degradation in both the capacity and the quality of an essential fish habitat: state of the Seine estuary (France) flatfish nurseries

The Bay of Seine is a potentially important nursery ground, especially for flatfish. This area, however, is also strongly anthropogenically influenced. A composite approach was chosen to compare the flatfish nursery function of the Seine estuary with other coastal and estuarine areas and to determine the impact of anthropogenic disturbances. It emerges that alteration of the nursery function in the Seine estuary is related to the loss of nursery habitat and to a lower quality of the residual surfaces. These observations stressed the importance of habitat destruction and decreased quality of remaining habitat for fish stocks renewal.     

Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.     

Identification of a transparent mutant tiger barb Puntius tetrazona and its use for in vivo observation of a Pleistophora sp. (Microsporidia) infection

A transparent mutant tiger barb Puntius tetrazona was identified and characterized by its transparent body, which allows clear visualization of internal organs. Hybridization of this mutant with the albino variant produces a transparent and albinoid double phenotype, and the transparency of this mutant is controlled by a recessive allele. Light microscopic and ultrastructural examinations show that in contrast to normal individuals, transparent mutants lack iridophores, and light penetrates unimpeded through the body. Pleistophora sp. infection was observed in vivo, allowing live observation of parasite dissemination and the consequent pathological alterations in the fish body as well as the simultaneous acquisition of data on the dynamics and spatial pattern of pathogenic invasion. It is superior to common fish models, as dynamic experimental data can be obtained from individual fish.     

Construction of a dual affinity tagged allele of the Rb1 tumor suppressor gene in the mouse

Loss of Rb1 tumor suppressor gene function is involved in the genesis of most human cancers. Novel therapies targeting Rb1 have been slow to develop because of our incomplete understanding of its molecular mechanisms of action. Rb1 protein (pRb) binds a host of cellular genes and proteins, and these molecular interactions mediate its various functions. Given the potential complexity of these molecular interactions and the lack of established methods for pRb purification, it has been difficult to systematically identify gene and protein interactions relevant to tumor suppression in different tissues in vivo. To address this limitation, we have generated a dual affinity tagged Rb1 allele in the mouse. The tagged allele functions as wild type and the encoded protein can be purified by tandem affinity chromatography. This allele will facilitate identification and characterization of native pRb molecular interactions in any tissue accessible in the mouse. genesis 48:121–126, 2010. © 2009 Wiley‐Liss, Inc.     

Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae.

Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin. One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted. Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures. Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2. These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.     

The ING1a Tumor Suppressor Regulates Endocytosis to Induce Cellular Senescence Via the Rb-E2F Pathway

The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated β-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57^KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.     

Oculocutaneous albinism in the i6 mutant of the medaka fish is associated with a deletion in the tyrosinase gene.

Three mutant alleles (i1, i4, and i5) of the tyrosinase gene in the i locus of the medaka fish Oryzias latipes have hitherto been described, all being associated with transposable element insertion. We have recently identified another allele causing a complete albino phenotype in homozygous carriers and named it i6. Sequence comparison between the tyrosinase gene for the i6 allele (Tyr-i6) and the wild-type gene previously obtained (Tyr-i+) revealed three deletions of 8, 44, and 245 bp. The first two deletions reside in an intron and are differences in the number of tandem tetranucleotide repeats that are polymorphic even among wild-type genes, and, thus, not likely to be responsible for the i6 albino phenotype. The largest deletion spans over the last 180 bp of the second intron and the first 65 bp of the third exon. Because of this deletion, the Tyr-i6 gene lacks the branch point sequence and the acceptor site for the second intron, both being considered to be necessary for normal RNA splicing. Therefore, the 245-bp deletion is likely to be responsible for the albino phenotype. With a mutant gene of this type, unlike ones bearing transposable element insertions, the possibility of reversion mutations to the wild-type would be negligible. Therefore, fish having the i6/i6 genotype should serve as superior recipients for the tyrosinase gene in rescue experiments.     

Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.     

Peculiarities of the lysozyme content in cods (order Gadiformes) and flatfish (order Pleuronectiformes)

There was analyzed the content of lysozyme-the parameter of the non-specific immunity-in kidney, liver, spleen, and blood serum in 8 fish species of orders Gadiformes and Pleuronectiformes. The cods are characterized by very low level of the enzyme or by the absence of its activity in some organs and in blood serum regardless of the fish habitat. The flatfish demonstrated diversity in the content and distribution of lysozyme in the body of fish of different species. Two flatfish species inhabiting the Okhotsk Sea, with the unique distribution of the enzyme, are revealed. Their peculiarity is due, first, to the highest content of lysozyme in the blood serum among all fish studied earlier; second, to exceeding 2–3 times the enzyme amount in blood serum, as compared to the studied immunocompetent organs. The obtained results confirm the concept that among the fish there exist different related groups differing by the lysozyme activity, and, possibly, by peculiarities of the non-specific immunity as a whole.