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.     

The primary structure of human chromogranin A and pancreastatin

A full-length clone encoding human chromogranin A has been isolated from a lambda gt10 cDNA library of a human pheochromocytoma. The nucleotide sequence reveals that human chromogranin A is a 439-residue protein preceded by an 18-residue signal peptide. Comparison of the protein sequence of human chromogranin A with that of bovine chromogranin A shows high conservation of the NH2-terminal and COOH-terminal domains as well as the potential dibasic cleavage sites, whereas the middle portion shows remarkable sequence variation (36%). This part of human chromogranin A contains a sequence homologous to porcine pancreastatin at residues 250-301. The sequence variation in this part of human chromogranin A compared to porcine pancreastatin is 32% and thus of the same magnitude as that between human and bovine chromogranin A. Therefore, the difference between porcine pancreastatin and the corresponding portions of bovine or human chromogranin A can be explained by species variation, suggesting that pancreastatin is derived from chromogranin A itself rather than a protein that is only similar to chromogranin A. Moreover, the pancreastatin sequence contained in human chromogranin A is flanked by sites for proteolytic processing. Together, these observations suggest that human chromogranin A may be the precursor for a human pancreastatin molecule and possibly for other, as yet unidentified, biologically active peptides.     

Gene response upon illumination in forming mRNA encoding peroxisomal glycollate oxidase

Glycollate oxidase is a constituent of leaf peroxisomes. Its biosynthesis is, like the biosynthesis of many chloroplastic proteins, controlled by light, via phytochrome. The level of mRNA coding for glycollate oxidase was determined at different stages of greening of etiolated plant cells. The appearance of glycollate oxidase mRNA in the cytoplasm was measured by hybridization with cDNA containing part of the coding sequence for glycollate oxidase. cDNA was prepared from enriched mRNA, inserted into the Pst I site of pBR 322, and cloned in Escherichia coli DH-1. By differential colony hybridization and hybrid selection, a clone containing a 670 bp sequence complementary to mRNA encoding glycollate oxidase was selected and identified. Northern blot hybridization was used to investigate mRNA levels induced by light. It was found that continuous light affected the formation of glycollate oxidase mRNA. When a large population of microbodies was present in the cells being induced, the immediate mRNA increase was very pronounced, and was detectable as little as 20 min after the beginning of the light treatment. In contrast, a lag period in the mRNA increase was observed when the induction was performed with etiolated leaves which are characterized by the occurrence of a rather small population of microbodies. For comparison, we measured the time-course of formation of mRNA coding for a light-induced chloroplastic protein, i.e., a protein of the light-harvesting complex. The time-courses of levels of the two mRNAs indicate that the program of gene expression differs between the two particular proteins destined either for chloroplasts or for peroxisomes. The formation of glycollate oxidase mRNA could also be stimulated by a short pulse of light, a treatment of 15 s being a sufficient trigger.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.     

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.     

Synthesis and degradation of aflatoxins by Aspergillus parasiticus. II. Comparative toxicity and mutagenicity of aflatoxin B1 and its autolytic breakdown products

A crude mycelial protein extract from a 16-day-old culture of A. parasiticus, on purification, lost 50% of its ability to degrade aflatoxin B1. The addition of hydrogen peroxide increased this activity to 97% of that of the crude extract. Ducklings dosed orally with aflatoxin extracts from 14- and 20-day-old cultures containing 46 micrograms or more of aflatoxin B1 developed enlarged livers, haemorrhaged and died in less than 10 days, giving and LD50 of 17.5 and 17.1 micrograms aflatoxin B1 per 50 g body weight respectively for each extract. When pure aflatoxin B1 was mixed with either the crude or purified mycelial protein extract the aflatoxin B1 level was decreased by 29% as was the toxicity of the mixture. The main breakdown product of aflatoxin B1 was isolated and was shown to have an RF value of 0.34, was non-fluorescent, and was non-toxic for ducklings at oral doses as high as 400 micrograms per 50 g body weight. The mutagenic effect of aflatoxin B1 on Salmonella typhimurium was relative to its concentration. The main breakdown product of aflatoxin B1 was non-mutagenic.     

Differences in regional capillary distribution and myocyte sizes in normal and hypertrophic rat hearts.

Data are reported which show significant regional capillary differences in left ventricular endocardium and epicardium of normal rats and of rats with hyperthyroid-induced cardiac hypertrophy. The epicardial region of control rats has 38% more capillaries than the endocardial region. Control endocardial myocytes are 62% larger in cross-sectional area than epicardial myocytes. Hypertrophic hearts exhibit regional differences in capillary density similar to those in the normal hearts, but there is an overall reduction of 12 and 17.5% in capillary density in both regions. The average cross-sectional area of myocytes increases 34.5% in the epicardium and 22.5% in the endocardium.     

The granin (chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.     

Localized increases in ovarian vascular permeability and leucocyte accumulation after induced ovulation in rabbits.

Colloidal carbon was injected i.v. in mature virgin rabbits at different times after induction of ovulation by human chorionic gonadotrophin (hCG, 100 iu) or mating. Before induction of ovulation, slight carbon leakage was observed in the inner vascular ring of the theca interna of antral follicles, but blood vessels in the other ovarian compartments were unstained. Between 4 and 10.5 h after hCG-treatment or mating, vascular leakage was most marked in the blood vessels of the interstitial gland and in the theca interna of antral follicles. Just before ovulation, carbon particles were observed between granulosa cells and some carbon was seeping into the follicular fluid of preruptured follicles. Vascular leakage was also observed over the follicle dome before rupture as well as at the dorsomedial junction between the mesovarium and the ovary. The blood vessels stained with carbon were 7-70 microns diameter, representing capillaries and postcapillary venules. About 6 h after hCG injection, an increased number of polymorphonuclear leucocytes migrated from the vessels of these ovarian compartments into the surrounding interstitial tissue. The number of leucocytes seen in the follicular wall and ovarian medulla increased markedly towards ovulation. During early corpus luteum formation, the number of leucocytes decreased markedly. The localized vascular changes seen after mating and hCG stimulation were similar to an inflammatory reaction and could form the basis for the formation of peritoneal exudate after ovulation in rabbits and periovulatory ascitic accumulation seen in the peritoneal cavity of women during the menstrual cycle.