Genomic signatures of breast cancer metastasis.

Despite significant advances in the treatment of primary cancer, the ability to predict the metastatic behavior of a patient's cancer, as well as to detect and eradicate such recurrences, remain major clinical challenges in oncology. While many potential molecular biomarkers have been identified and tested previously, none have greatly improved the accuracy of specimen evaluation over routine histopathological criteria and they predict individual outcomes poorly. However, the recent introduction of high-throughput microarray technology has opened new avenues in genomic investigation of cancer, and through application in tissue-based studies and appropriate animal models, has facilitated the identification of gene expression signatures that are associated with the lethal progression of breast cancer. The use of these approaches has the potential to greatly impact our knowledge of tumor biology, to provide efficient biomarkers, and enable development towards customized prognostication and therapies for the individual.

Comparison of telomerase and CD44 expression as diagnostic tumor markers in lesions of the thyroid.

The analysis of the tissue expression patterns of both the telomerase enzyme and the adhesion molecule CD44 has highlighted these molecules as potential tumor markers. In this study, the expression of these markers was analyzed in frozen tissue samples of the same human thyroid lesions, and the data were compared to evaluate their application to tumor diagnosis. The study analyzed 12 malignant specimens, including 5 papillary, 3 follicular, 2 anaplastic, 1 medullary, and 1 low-grade Hurthle cell carcinoma and 17 specimens from benign lesions, including cases of adenoma, hyperplasia, and Graves' disease. Telomerase expression was analyzed by assay of enzyme activity using the telomeric repeat amplification protocol and by reverse transcription-PCR detection of human telomerase reverse transcriptase (hTERT) mRNA. Nine of 12 (75%) malignant samples and the two Graves' disease samples were evaluated as positive for telomerase activity by the telomeric repeat amplification protocol assay. The presence of hTERT mRNA was detected in 8 (67%) of 12 malignant tissues and in 5 (29%) of 17 benign thyroid tissue samples. The expression of CD44 transcripts containing variant exons 7, 8, and 11 was evaluated by reverse transcription-PCR/Southern blot analysis. Of the 12 malignant samples, 9 (75%) included transcripts containing exon 7, 10 (83%) included transcripts containing exon 11, and 11 (92%) included transcripts containing exon 8. However, these CD44 exons were also present in transcripts in a high proportion of benign samples. Five (28%), 10 (59%), and 6 (35%) benign samples contained CD44 transcripts, including variant exons 7, 8, and 11, respectively. The measurement of telomerase activity proved to be the most specific for the detection of thyroid carcinoma in frozen tissue samples as a single analyte, but diagnostic accuracy was increased by the combination of telomerase and CD44 analyses.

Telomerase activity in soft-tissue and bone sarcomas.

The telomerase enzyme is a reverse transcriptase capable of replacing the telomeric DNA sequences that are lost at each cell division. Telomerase activation permits extended cell proliferation beyond normal senescence checkpoints, and accordingly, telomerase activity has been detected in a wide range of malignant cells and tissues but is absent in terminally differentiated somatic cells. To date, the majority of cancer-related telomerase analyses have been performed on carcinomas that originate from epithelial cells, and few reports have included tumors originating from nonepithelial cells. In this study, we used the PCR-based telomeric repeat amplification protocol (TRAP) to assay telomerase activity in nuclear protein extracts obtained from a range of malignant and benign connective tissue lesions. In total, 62 histologically diagnosed specimens were analyzed including 37 sarcomas, 7 benign mesenchymal tumors, 12 normal tissue samples, and 6 carcinoma metastases obtained from bone. Thirty (81%) of the 37 primary sarcoma samples contained telomerase activity, and four of the six carcinoma metastases were also positive. Conversely, telomerase activity was detectable in only one of seven benign lesions and in none of the 12 normal connective tissue controls. Tumors of connective tissue origin can sometimes be difficult to categorize and to evaluate microscopically with regard to clinical management. As is the case in carcinomas, the presence of telomerase activity appears to be indicative of malignancy in mesenchymal tumor biopsy material and therefore may be useful as an adjunct to the pathologist in the assessment of borderline cases.

A novel pancreatic beta-cell isoform of calcium/calmodulin-dependent protein kinase II (beta 3 isoform) contains a proline-rich tandem repeat in the association domain.

There is evidence for a role for calcium/calmodulin-dependent protein phosphorylation in regulation of insulin secretion but the molecular nature of the kinase(s) responsible is unknown. In this study, the screening of a neonatal rat islet cDNA library resulted in the isolation of a 2 kb clone that was 99% homologous to the beta' isoform of calcium/calmodulin-dependent protein kinase II. The predicted 589 amino acid sequence with a calculated mass of 64,976 Da contained a 24 amino acid deletion in addition to the 15 amino acid deletion that differentiates the beta' from the beta isoform, and included an 86 amino acid novel domain consisting of a tandem repeat of proline-rich residues. The expression of this new isoform of calcium/calmodulin-dependent protein kinase II (beta 3) was confirmed in beta-cell lines and testis by DNA amplification of the sequence encoding the inserted domain by reverse transcriptase-polymerase chain reaction, followed by Southern analysis.

Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion.

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.

Selective modulation of P-glycoprotein's ATPase and anion efflux regulation activities with PKC alpha and PKC epsilon in Sf9 cells.

The modulation of P-glycoprotein's (Pgp) ATPase activity and its ability to regulate swelling-activated 125I efflux, by PKC alpha and PKC epsilon, was examined in insect cells. Recombinant baculovirus was used to express human Pgp in Sf9 cells and Pgp was also coexpressed with either PKC alpha or PKC epsilon. ATPase assays showed the enzyme activity of Pgp to be elevated during co-expression with the Ca2+ dependent isoform PKC alpha, but not with the Ca2+ independent variant PKC epsilon. Furthermore, neither isoform, when co-expressed with Pgp, altered the swelling-activated efflux of 125I from Sf9 cells. However, in cells co-expressing Pgp/PKC (alpha or epsilon), pre-treatment with the phorbol ester TPA significantly reduced the swelling-activated 125I efflux with both PKC isoforms. Our results suggest that phosphorylation with the Ca2+ independent variant PKC epsilon does not regulate the ATPase activity of Pgp and that stimulation of PKC with TPA alters the swelling-activated efflux of anions from insect cells expressing Pgp.

Molecular diagnostic trends in urological cancer: biomarkers for non-invasive diagnosis.

The early detection of urological cancers is pivotal for successful patient treatment and management. The development of molecular assays that can diagnose disease accurately, or that can augment current methods of evaluation, would be a significant advance. Ideally, such molecular assays would be applicable to non-invasively obtained body fluids, enabling not only diagnosis of at risk patients, but also asymptomatic screening, monitoring disease recurrence and response to treatment. The advent of advanced proteomics and genomics technologies and associated bioinformatics development is bringing these goals into focus. In this article we will discuss the promise of biomarkers in urinalysis for the detection and clinical evaluation of the major urological cancers, including bladder, kidney and prostate. The development of urine-based tests to detect urological cancers would be of tremendous benefit to both patients and the healthcare system.

Molecular epidemiology of human rotavirus infection in Coro, Venezuela.

From December 1984 to December 1987, rotaviruses were detected in 115 (24%) of 470 hospitalized children, by electrophoretic analysis of viral RNA. Three peaks of increased incidence were observed, coincident with the cooler months of each year. Rotavirus was found in 31% of the children with diarrhea and in 47% of those who were severely dehydrated. Vomiting was significantly associated with rotavirus shedding in diarrheic children. Eleven electropherotypes were identified, showing a sequential pattern of appearance. Strains exhibiting a "short" pattern were present in 10% of the positive cases. The rate of infection increased to 71% in march 1987, when a mixed infection with two genotypically different strains was found and when electropherotypes with an extra RNA segment appeared.

[The population and the environment]. / Población y medio ambiente.

This paper discusses, on the basis of the Bruntland Report on development and environment, the relationship between population and development possibilities, and its consequences on natural and man made resources. The author emphasizes the need to design development policies in which the protection of the environment is a priority, specially in Latin American countries.

Population and new international order: a missing link?

PIP: The author examines the relationship between population growth and policies and the formulation of the new international economic order. The implications of future population growth and the international distribution of population are discussed. Employment trends are studied with reference to labor migration, the international transfer of technology, and the status of women^ieng

Non-random reassortment between the tripartite RNA genomes of La Crosse and snowshoe hare viruses.

The process of reassortment between the tripartite RNA genomes (segments designated L, M and S) of snowshoe hare and La Crosse bunyaviruses (Bunyaviridae) has been investigated by polymerase chain reaction analysis of greater than 250 progeny recovered at 72 h post-infection from dual wild-type virus infections involving high multiplicities (approximately 5) of each virus in a BHK-21 cell line. Statistical analysis of the data indicated that RNA segment reassortment was not random, and for these two viruses the data appeared to fit the hypothesis that there was a preference for homologous L-M and M-S associations among the progeny formed.

Role of telomerase in cell senescence and oncogenesis.

The ends of linear chromosomes are capped by specialized nucleoprotein structures termed telomeres. Telomeres comprise tracts of noncoding hexanucleotide repeat sequences that, in combination with specific proteins, protect against degradation, rearrangement, and chromosomal fusion events. Due to the polarity of conventional DNA synthesis, a net loss of telomeric sequences occurs at each cell division. It has been proposed that this cumulative telomeric erosion is a limiting factor in replicative capacity and elicits a signal for the onset of cellular senescence. To proliferate beyond the senescent checkpoint, cells must restore telomere length. This can be achieved by telomerase, an enzyme with reverse-transcriptase activity. This enzyme is absent in differentiated somatic tissues, but telomerase reactivation has been detected in most tumors. Much investigative effort is focusing on telomere dynamics with a view to possible manipulation of cellular proliferative potential. In this article, we review the role of telomeres and telomerase in senescence and tumor progression, and we discuss the potential use of telomerase in diagnosis and treatment.

Telomerase in cancer: clinical applications.

The biology of telomeres and telomerase has been the subject of intensive investigative effort since it became evident that they play a significant role in two important biological processes, the loss of cellular replicative capacity inherent to organismal ageing and the unrestricted cell proliferation characteristic of carcinogenesis. Telomere shortening in normal cells is a result of DNA replication events, and reduction beyond a critical length is a signal for cellular senescence. One of the cellular mechanisms used to overcome proliferative restriction is the activation of the enzyme telomerase, which replaces the loss of telomeric DNA that occurs at each cell division. Studies have demonstrated that tumours have shorter telomeres than normal tissue and that telomerase is activated in up to 90% of all human cancers while it is present only in a limited range of normal adult tissues. The role of telomerase in the extension of the cellular replicative lifespan has recently been shown by ectopic expression of the enzyme, being consistent with the oncogenesis model whereby the acquisition of an 'immortal' phenotype is a requirement for advanced tumour progression. In this article we review the present knowledge of telomeres and telomerase in cancer and discuss the potential use of this enzyme as a diagnostic and prognostic tumour marker and as a target for cancer therapy.

Protein synthesis in cells infected with bovine rotavirus.

Bovine rotavirus was found to multiply efficiently in LLC-MK2 cells, a continuous line of rhesus monkey kidney, with a growth cycle which was essentially completed within 9 h after infection. The presence of low concentrations of trypsin (10 microgram/ml) in the virus inoculum was essential for infectivity. Polyacrylamide gel electrophoresis of infected cell extracts demonstrated the synthesis of at least eight virus-specific polypeptides 6 h post-infection with mol. wt. ranging from 102 X 10(3) to 29 X 10(3). Six polypeptides (about p102K, p91K, p84K, p37K and p34K) were identified as structural components of the virion. Two other polypeptides (54K and p29K) were identified as non-structural components. The synthesis of non-structural polypeptides appeared to precede that of the structural proteins. Pulse-chase experiments showed only one minor post-translation modification of the virus-specified proteins, namely an increase in the mobility of the 29K polypeptide.

CD44 cell adhesion molecules.

The CD44 proteins form a ubiquitously expressed family of cell surface adhesion molecules involved in cell-cell and cell-matrix interactions. The multiple protein isoforms are encoded by a single gene by alternative splicing and are further modified by a range of post-translational modifications. CD44 proteins are single chain molecules comprising an N-terminal extracellular domain, a membrane proximal region, a transmembrane domain, and a cytoplasmic tail. The CD44 gene has only been detected in higher organisms and the amino acid sequence of most of the molecule is highly conserved between mammalian species. The principal ligand of CD44 is hyaluronic acid, an integral component of the extracellular matrix. Other CD44 ligands include osteopontin, serglycin, collagens, fibronectin, and laminin. The major physiological role of CD44 is to maintain organ and tissue structure via cell-cell and cell-matrix adhesion, but certain variant isoforms can also mediate lymphocyte activation and homing, and the presentation of chemical factors and hormones. Increased interest has been directed at the characterisation of this molecule since it was observed that expression of multiple CD44 isoforms is greatly upregulated in neoplasia. CD44, particularly its variants, may be useful as a diagnostic or prognostic marker of malignancy and, in at least some human cancers, it may be a potential target for cancer therapy. This review describes the structure of the CD44 gene and discusses some of its roles in physiological and pathological processes.

The design and use of specific genetic probes to identify closely related bunyaviruses and to determine the genotype of their recombinants.

Viruses that are very closely related to each other at the genetic and gene product level can prove difficult to distinguish, although they may differ in phenotype (for example in their virulence or vector preferences). A chimeric genetic probe has been developed and tested to distinguish the S RNAs of two closely related bunyaviruses, snowshoe hare and La Crosse viruses. The technique is applicable to other RNA species of these two bunyaviruses.

PTEN expression is maintained in sporadic colorectal tumours.

Loss of PTEN (phosphatase and tensin homologue deleted from chromosome 10) function has been implicated in the progression of several types of cancer. Allele loss close to the PTEN locus occurs in sporadic colon cancer and germline PTEN mutations cause Cowden disease, an inherited cancer syndrome characterized by an increased incidence of gastrointestinal tract lesions that can progress to colorectal carcinoma. However, although PTEN is a good candidate for involvement in the pathogenesis of sporadic colon cancer, previous analyses have not revealed a high frequency of somatic mutations in colorectal tumours. Alternative mechanisms which could lead to a loss of PTEN expression in colon cancer have not been investigated. This study monitored PTEN mRNA and protein levels in a panel of 50 tumour tissues obtained from 35 patients with sporadic colon cancer. RT-PCR and immunohistochemistry were used to evaluate the expression of mRNA and protein, respectively, in normal, adenoma and adenocarcinoma colorectal tissues as well as in metastatic lesions. To overcome the problem of heterogeneity and normal stromal cell contamination in homogenized tissue specimens, specific cell types were isolated by microdissection prior to PCR analysis. No loss of PTEN expression was evident in any of the colon tissues examined. PTEN protein was localized exclusively in the cytoplasm of normal and tumour cells and no correlation of immunostaining intensity and tumour stage or grade was revealed. As with previous deletion and mutation analyses, the present study suggests that loss of PTEN expression is not prevalent in sporadic colon cancer.