Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Wnt Pathway Activation Increases Hypoxia Tolerance during Development

Adaptation to hypoxia, defined as a condition of inadequate oxygen supply, has enabled humans to successfully colonize high altitude regions. The mechanisms attempted by organisms to cope with short-term hypoxia include increased ATP production via anaerobic respiration and stabilization of Hypoxia Inducible Factor 1α (HIF-1α). However, less is known about the means through which populations adapt to chronic hypoxia during the process of development within a life time or over generations. Here we show that signaling via the highly conserved Wnt pathway impacts the ability of Drosophila melanogaster to complete its life cycle under hypoxia. We identify this pathway through analyses of genome sequencing and gene expression of a Drosophila melanogaster population adapted over >180 generations to tolerate a concentration of 3.5–4% O₂ in air. We then show that genetic activation of the Wnt canonical pathway leads to increased rates of adult eclosion in low O₂. Our results indicate that a previously unsuspected major developmental pathway, Wnt, plays a significant role in hypoxia tolerance.     

Seasonal Variation of Western White Pine (Pinus monticola D. Don) Foliage Proteins

Recently, a western white pine protein, Pin m III, was shownto be associated with overwintering and frost hardiness of westernwhite pine foliage. To examine whether Pin m III is directlyinvolved in frost hardiness by functioning as an antifreezeprotein, work is underway to clone the gene encoding this proteinand to assess the function of this gene in freezing toleranceby incorporating the gene in a test plant, such as tobacco.Here, we examined in more detail, by SDS-PAGE and also by twodimensional gel electrophoresis, the seasonal variation of additionalproteins in western pine foliage. SDS-PAGE analysis of threeseedlots showed that different proteins reached a maximum levelin different months, although most proteins (5 to 11) reacheda maximum level in winter months (December, January and February).The 2-D gel analysis of foliage sampled on three harvest dates(October, January and April) of one seedlot revealed a seasonalvariation of a large number proteins (76 to 184). Of the seasonallyvaried proteins, the amino terminal sequence of several proteinsincluding Pin m III was determined. One of the sequences wasidentified by homology to that of the small subunit of ribulosebiphosphate carboxylase, whose level increased substantiallyfrom fall to spring. The amino terminal sequence of Pin m IIIhad 89% homology to a sugar pine protein, Pin 11. The anti-photosystemII antibody was used to monitor the annual variation of theextrinsic 23-kDa photosystem II protein. The level of the extrinsic23-kDa photosystem II protein decreased slowly as fall progressedand reached its lowest level in December and then increasedin early spring indicating that this variation is due to photosyntheticactivity of the foliage during the season. (Received July 29, 1995; Accepted March 5, 1996)     

Secretion of β-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

Summary A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of -lactamase and determine optimal conditions for its secretion into the culture media.     

Macro- and microscopic morphology of the flank scales of families Lutjanidae and Serranidae from the Persian Gulf Coral Reefs (Teleosts: Perciformes)

Macro- and microscopic characteristics of flank scales for 12 species were investigated from the Persian Gulf Coral Reefs. In Lutjanus argentimaculatus and L. russellii (family Lutjanidae), the scales of different flank regions were not different, while four characters showed variation in the scale of L. lutjanus i.e., scale shape (pentagonal, hexagonal and square), anterior margin (waved, scalloped and smooth), focus shape (circular and oblong) and focus position (postero-central and central), displayed variation. Scale type (ctenoid) and posterior margin (transforming ctenii) did not show variation and could be considered to be specific in this family. In Epinephelus chlorostigma (family Serranidae), the scales of flank regions did not display variation, while in E. areolatus, E. diacanthus and E. radiates, the scales showed considerable variation. The most variable characters were scale shape, posterior margin and focus shape. Therefore, in fish systematics studies on the base of scale, it is particularly important to compare scales from the same flank regions. Also, some criteria such as size-dependent alternation, ontogenetic changes and variation between flank regions, should be considered. This study supports the potential of scale morphology to help for the understanding of fish diversity in the coral reef ecosystem.     

Extracellular addition of a domain of HIV-1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death

Vpr is a virion-associated protein of human immuno-deficiency virus type 1 (HIV-1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell-growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by-stander cells during AIDS.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Intermolecular base-paired interaction between complementary sequences present near the 3'' ends of 5S rRNA and 18S (16S) rRNA might be involved in the reversible association of ribosomal subunits.

Highly conserved sequences present at an identical position near the 3' ends of eukaryotic and prokaryotic 5S rRNAs are complementary to the 5' strand of the m2(6)A hairpin structure near the 3' ends of 18S rRNA and 16S rRNA, respectively. The extent of base-pairing and the calculated stabilities of the hybrids that can be constructed between 5S rRNAs and the small ribosomal subunit RNAs are greater than most, if not all, RNA-RNA interactions that have been implicated in protein synthesis. The existence of complementary sequences in 5S rRNA and small ribosomal subunit RNA, along with the previous observation that there is very efficient and selective hybridization in vitro between 5S and 18S rRNA, suggests that base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small ribosomal subunit might be involved in the reversible association of ribosomal subunits. Structural and functional evidence supporting this hypothesis is discussed.     

Trisomic analysis of ribosomal RNA cistron multiplicity in barley (Hordeum vulgare L.)

Ribosomal RNA cistron numbers in all the seven primary trisomics of diploid barley (Hordeum vulgare L.) were determined by DNA-rRNA filter hybridisation. Trisomies for the nucleolus organiser (NO) chromosomes 6 and 7 showed the highest levels of rDNA (DNA complementary to rRNA) indicating the localisation of rRNA cistrons on the NOs. Chromosomes 6 and 7 possessed 1,580 and 2,690 rRNA (18S + 5.8S + 26S) cistrons respectively. Trisomics for the other chromosomes (except for 3) also displayed levels of rDNA significantly higher (22–32%) than the diploid controls although the dosage of NOs was not altered. These non-specific increases were also present in trisomics for 6 and 7 (NOs) which showed further increases equivalent to their respective contributions. The nonspecific increases due to trisomy is indicative of rDNA compensation. Such increases did not persist in diploid sibs of the trisomics, demonstrating the nonheritable nature of the compensation.