Tissue invasion and metastasis: Molecular, biological and clinical perspectives.

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), ß-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-ß), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.

Differential expression of Glut 1 mRNA and protein levels correlates with increased sensitivity to the glyco-conjugated nitric oxide donor (2-glu-SNAP) in different tumor cell types.

Nitric Oxide (NO) releasing agents can serve as potent cytotoxic agents. However at present there are no effective ways to target delivery of NO donors like S-nitroso-N-acetyl-penicillamine (SNAP). SNAP conjugated to glucose (2-gluSNAP) can be readily transported across the membrane by GLUT 1 transporters. Therefore, sensitivity of cells to 2-gluSNAP may depend on glucose-transporter GLUT 1. We evaluated the cytotoxicity of SNAP and 2-gluSNAP on a GLUT 1 rich glioblastoma cell line T98G and GLUT 1 deficient osteoblastoma cell line 143B and its mitochondria-deficient variant rhoo (cell line 206). The cytotoxity of SNAP and 2-gluSNAP was assessed by clonogenic assay performed in the above cell lines in vitro. Immunoblotting and semi-quantitative real-time PCR assays were used to evaluate the expression of GLUT 1 transporter at protein and mRNA levels. The glioblastoma cell line T98G was more sensitive to 2-gluSNAP than unconjugated SNAP. SNAP and 2-gluSNAP affected the osteosarcoma cell lines 143B and rhoo poorly. Immunoblot analysis detected GLUT 1 protein in T98G cells and not in 143B or rhoo. There was about a 10-fold difference in GLUT 1 mRNA level in T98G cells compared to 143B and rhoo cell lines. This is consistent with our cytotoxicity studies and immunoblot analysis. Our results give credence to our hypothesis that the sensitivity to NO donors can be increased by glyco-conjugation and the cytotoxicity of the glyco-conjugated NO donors depends on the expression of GLUT 1 mRNA and protein.

A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of sigma(s).

The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or sigma(S)) required for the expression of stationary-phase genes. In the first published rpoS sequence from E. coli K-12 codon 33 is given as CAG. However, several subsequent independent studies found the amber codon TAG at this position ( rpoSAm). Besides this amber codon, other codons such as TAT have also been found at this location in rpoS. Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E. coli K-12. Five different stocks of the strain W3110, which differ in the levels of sigma(S) protein they express, were investigated. We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five. W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants. Such rpoSAm mutants would be expected to show no RpoS activity. The retention of partial or intermediate sigma(S) activity by suppressor-free rpoSAm mutants is therefore puzzling. We propose that a functional, N-terminally truncated, sigma(S) (Delta1-53sigma(S)) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33. It has recently been reported that a fragment of RpoS (Delta1-53sigma(S)) that lacks the first 53 amino acids is functional when synthesized in vivo. Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E. coli K-12 strains is the amber codon TAG.

In vitro properties of RpoS (sigma(S)) mutants of Escherichia coli with postulated N-terminal subregion 1.1 or C-terminal region 4 deleted.

Derivatives of the stationary-phase sigma factor sigma(S) of Escherichia coli lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several sigma(S)-dependent promoters on supercoiled DNA templates. Unlike wild-type sigma(S), however, the deletion derivatives were inactive on relaxed templates. The anomalous slow electrophoretic mobility of sigma(S) on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in sigma(S) may be structurally and functionally analogous to subregion 1.1 of sigma(70), substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of sigma(70) (V. Gopal and D. Chatterji, Eur. J. Biochem. 244:614-618, 1997).

Escherichia coli RNase M is a multiply altered form of RNase I.

RNase M, an enzyme previously purified to homogeneity from Escherichia coli, was suggested to be the RNase responsible for mRNA degradation in this bacterium. Although related to the endoribonuclease, RNase I, its distinct properties led to the conclusion that RNase M was a second, low molecular mass, broad specificity endoribonuclease present in E. coli. However, based on sequence analysis, southern hybridization, and enzyme activity, we show that RNase M is, in fact, a multiply altered form of RNase I. In addition to three amino acid substitutions that confer the properties of RNase M on the mutated RNase I, the protein is synthesized from an rna gene that contains a UGA nonsense codon at position 5, apparently as a result of a low level of readthrough. We also suggest that RNase M is just one of several previously described endoribonuclease activities that are actually manifestations of RNase I.

Report on a patient with paroxysmal cold hemoglobinuria.

Antibodies against the human blood group P antigen (anti-P alloantibodies) agglutinate phenotype P1 and P2 erythrocytes treated with papain at 4 degrees C but not phenotype PK and p erythrocytes. This condition is referred to as an autoimmune disease of paroxysmal cold hemoglobinuria (PCH), and the anti-P specificity is found in the cold hemagglutinins. Serum from a patient suspected to be suffering from PCH by the cold auto-agglutination properties was tested for anti-P specificity, using papain-treated O blood group erythrocytes. The serum-mediated hemagglutination and the serum and complement-mediated immunohemolysis were inhibited by globoside (P antigen GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide) and Forssman glycosphingolipid (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide). Therefore, we concluded that she had PCH. She was completely cured 6 months later.

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost, Heteropneustes fossilis.

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleost Heteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9-34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.

Transfer RNA analysis during the reproductive cycle of a freshwater teleost, H. fossilis.

Transfer RNA was analyzed qualitatively as well as quantitatively from ovaries of the fresh water teleost Heteropneustes fossilis for twelve months. The tRNA samples were found to be pure and devoid of any high molecular weight RNA or DNA contaminations. The quantity of tRNA as well as its biological activity, assayed by in vitro aminoacylation using homologous aminoacyl tRNA synthetases, were found to be higher during resting and preparatory (pre-vitellogenic) phases, i.e. from November to March, as compared to vitellogenic and spawning phases of the fish, i.e. from April to October. The highest tRNA pool and its activity was found in the month of February, which coincides with the early preparatory phase. The results indicate that the accumulation of active tRNA starts in the resting phase. Such an accumulation of tRNA may be a part of the enrichment of mature eggs with complete translational machinery before ovulation in order to cope with the high rate of protein synthesis after fertilization.