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.     

Allosteric interference in oncogenic FLI1 and ERG transactions by mithramycins

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Distribution of zinc fractions and their transformation in submerged rice soils

Distribution of different forms of Zn in 16 acid alluvial rice growing soils of West Bengal (India) and their transformation on submergence were studied. The results showed that more than 84% of total Zn occurred in the relatively inactive clay lattice-bound form while a smaller fractionviz. 1.1, 1.6, 11.1 and 2.0 per cent of the total occurred as water-soluble plus exchangeable, organic complexed, amorphous sesquioxide-bound and crystalline sesquioxide bound forms, respectively. All these four Zn forms showed significant negative correlations with soil pH (r=−0.48^**, −0.39^*, −0.61^** and −0.67^**, respectively), while the latter two Zn forms showed significant positive correlations with Fe₂O₃ (0.68^** and 0.88^***) and Al₂O₃ (0.89^*** and 0.75^***) content of the soils. The different Zn forms were found to have positive and significant correlations amongst each other, suggesting the existence of a dynamic equilibrium of these forms in soil. Submergence caused an increase in the amorphous sesquioxide-bound form of Zn and a decrease in each of the other three forms. The magnitude of such decreases in water-soluble plus exchangeable and crystalline sesquioxide-bound forms was found to be correlated negatively with initial pH values of the soils and positively with the increase in the amorphous sesquioxide-bound form, indicating their adsorption on the surface of the freshly formed hydrated oxides of Fe, which view was supported by the existence of significant positive correlation between the increase in the amorphous sesquioxide-bound form of Zn and that in AlCl₃-extractable iron. The existence of a positive correlation between the decrease in crystalline sesquioxide-bound Zn and that in Fe₂O₃ content in soil suggested that on waterlogging the soil Zn occluded in the cry talline sesquioxide was released as a result of reduction of Fe₂O₃.     

Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

The plasmid pEAP31 contains an alkaliphilic-Bacillus penicillinase gene and a colicin E1 kil gene. Escherichia coli HB101 carrying pEAP31 grown at high temperature released outer-membrane proteins, lipopolysaccharide and phosphatidylethanolamine into the culture medium. Concurrently, penicillinase that had accumulated in the periplasm of the organism was released from the cells. Phospholipase A1-A2 in the outer membrane was not activated in the organism. The results suggest that the release of accumulated periplasmic penicillinase from the producer cells was caused by partial disruption of the outer membrane mediated by the Kil peptide.     

Cadmium resistance in some members of Enterobacteriaceae

Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16), and Serratia marcescens (16) were screened for Cd resistance or sensitivity. Only one strain each of these was resistant to high levels (25 n moles/0.05 ml) CdCl2. The Minimal inhibitory concentration (MIC) of sensitive strains ranged from 0.8-5 micrograms/ml. All the resistant strains were simultaneously resistant to a number of antibiotics. Treatment with sodium dodecyl sulfate eliminated resistance to Cd and to some antibiotics.     

A study of the hydration and thermodynamics of warm-water and cold-water fish collagens.

The hydrated volumes, Vh, of collagens extracted from various fish species were calculated by using the Simha-Einstein equation, and it was found that the hydration of warm-water fish collagen is greater than that of cold-water fish collagen (halibut). Although the intrinsic viscosities of warm-water fish (bigeye-tuna, carp and catfish) collagens are almost the same, the hydrated volume of bigeye-tuna collagen is approx. 1.5 and 3 times those of carp and catfish collagens respectively. The extent of hydration at 20 degrees C is in the following order: bigeye tuna greater than carp greater than catfish greater than halibut. The various thermodynamic activation parameters (delta G*, delta H* and delta S*) were calculated and it was found that they are useful for determining the exact denaturation temperature. It was calculated that the denaturation temperatures of halibut, bigeye-tuna, carp and catfish collagens are 17, 31, 32 and 26-30 degrees C respectively. The variations of hydration, intrinsic viscosity, denaturation temperature and the thermodynamic parameters with the variation of concentration of catfish collagen were also thoroughly examined. The change of thermodynamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results.     

Purification and partial characterization of two azoreductases from Shigella dysenteriae Type 1

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).