Brigitte Marian

Cells obtained from colorectal microadenomas mirror early premalignant growth patterns in vitro

LT97, a permanent cell line consisting of epithelial cells with an early premalignant genotype was established from small colorectal polyps. LT97 cells have lost both alleles of the APC tumour suppressor gene. In addition, they carry a mutated Ki-ras oncogene, while TP53 is normal. LT97 growth characteristics are thus representative of early adenomas. They had to be passaged as multicellular aggregates indicating a dependency of survival on cell-cell contact and in accordance with their premalignant genotype were not capable of growth in soft agar. LT97 cells did express both the EGF-receptor and small amounts of TGF(alpha) establishing an autocrine growth or survival pathway. However, in spite of autocrine TGF(alpha) production, growth was strongly dependent on exogenous growth factors--mainly EGF, insulin and HGF. Inhibition of the EGF-receptor kinase induced apoptosis at an IC(50) concentration of 4 micromolar indicating that TGF(alpha) activated survival pathways in the early adenoma cell.

Induction of lipid peroxidation in biomembranes by dietary oil components

Prooxidant formation and resulting lipid peroxidation are supposed to be involved in the pathogenesis of various diseases including cancer. Cancer risk is possibly influenced by the composition of diet with high intake of fat and red meat being harmful and high consumption of fruits and vegetables being protective. Since dietary oils may contain potential prooxidants, the aim of the present study was to prove (i) whether oxidative stress in biomembranes may be induced by dietary oils and if, (ii) which impact it has on the viability and proliferation of cultured colon (carcinoma) cells. Lipid hydroperoxide content in dietary oils increased after heating. Linoleic acid hydroperoxide (LOOH) and/or oils with different hydroperoxide contents induced lipid peroxidation in liposomes, erythrocyte ghosts and colon cells. Upon incubation with liposomes, both LOOH and heated oil induced lipid peroxidation only in the presence of iron and ascorbate. LOOH was sufficient to start lipid peroxidation of erythrocyte ghosts. LOOH incorporates into the lipid bilayer decreasing membrane fluidity and initiating lipid peroxidation in the lipid phase. When cultured cells (IEC18 intestinal epithelial cells, SW480 and HT29/HI1 colon carcinoma cells) were exposed to LOOH, they responded by cell death both via apoptosis and necrosis. Cells with higher degree of membrane unsaturation were more susceptible and antioxidants (vitamin E and selenite) were protective indicating the involvement of oxidative stress. Thus, peroxidation of biomembranes can be initiated by lipid hydroperoxides from heated oils. Dietary consumption of heated oils may lead to oxidative damage and to cell death in the colon. This may contribute to the enhanced risk of colon cancer due to regenerative cell proliferation.

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.

Flavaglines: a group of efficient growth inhibitors block cell cycle progression and induce apoptosis in colorectal cancer cells.

Flavaglines are flavonol–cinnamate‐derived cyclopenta[b]benzofurans, so far reported only for the genus Aglaia of the plant family Meliaceae. They represent a group of highly bioactive metabolites already known for their strong antileukemic activities. To assess their suitability as chemotherapeutic drugs in colorectal cancer, their cytostatic effects and the underlying mechanisms of action were analyzed in colorectal tumor cell lines. Aglaiastatin was the most active flavagline, inhibiting growth and inducing apoptosis at nanomolar concentrations in SW480 and HT29/HI1 carcinoma cells, while the premalignant adenoma cell lines VACO235 and LT97 as well as the normal intestinal epithelial cell line IEC18 were 1,000 times less sensitive (IC50 > 10 μM). In SW480 cells, aglaiastatin caused cell cycle block in early mitosis, demonstrated by a shift of cell cycle distribution 24 and 48 hr after addition of aglaiastatin and by an increased content of cyclin B after 6 hr together with a decreased level of cyclin A as early as 2 hr after exposure. In addition, induction of apoptosis could be shown by the characteristic morphology of apoptotic nuclei, loss of MMP and downmodulation of bclxl. Strong activation of p38 was observed after 2 hr of exposure, indicating that apoptosis may be induced via a p38‐mediated stress pathway.

Apoptosis in human colorectal tumours: ultrastructure and quantitative studies on tissue localization and association with bak expression

Abstract Apoptotic cell death in human tumours has been demonstrated by electron and light microscopy. In adenomas, fragmented and apoptotic nuclei and signs of phagocytosis have been observed close to the basement membrane. In carcinomas the characteristic structures were apoptotic bodies with small fragments of chromatin. DNA fragmentation was shown by in situ end-labelling. Quantitative assessment of apoptosis and proliferation revealed a high apoptotic index (AI) in all types of adenoma (tubular: 1.77±0.35%, tubulovillous: 2.38± 0.41%; villous: 3.3±0.39%) as well as loss of compartmentalization of proliferating and dying cells. In carcinomas a shift towards proliferation was evident, as shown by lower AIs than in adenomas (0.9±0.68% and 1.1±0.12% for moderately and poorly differentiated tumours), higher Ki67 indices (38.32±2.23% and 57± 3.89%, respectively) and higher mitosis (0.9±0.56% and 1.21±0.17%, respectively). However, apoptosis was observed in all tumours and is available as a target for therapeutic intervention. Expression of the apoptosis related proteins bcl-2 and bak also reflected loss of compartmentalization. While bcl-2 did not show a consistent relationship to AI in tumour specimens, bak was positively correlated with apoptosis in 4 of 8 adenomas and 4 of 7 carcinomas, suggesting a role for this protein in the induction of apoptosis in a subset of tumours.

Survival of normal colonic epithelial cells from both rats and humans is prolonged by coculture with rat embryo colonic fibroblasts

Primary cultures of normal colonic epithelial cells from both humans (HCEC) and rats (RCEC) have been established using coculture with colon fibroblasts isolated from rat term embryos. While no other factors we have analyzed had any effect on the survival of epithelial cells, which is normally 3–4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell–cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology and forming microvilli. DNA synthesis as measured by BrdU uptake into DNA varied widely between colonies of the same culture depending on cell morphology: flat colonies of RCECs contained 5.7%±0.56% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3%±2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0±1.0 and 23.0±2.0 colonies/well respectively; p≤0.05 increased above control) and size (1022±155 and 1207±158 cells/colony respectively; p≤0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2%±3.9% in controls to 21.3%±3.8% in the EGF/FGF group (p≤0.05) owing to the high proportion of flat colonies consisting of resting cells.The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.

Modification of histones during the mitotic and meiotic cycle of yeast

Core histones (H2a ,H2b ,H3 ,H4) of yeast have been shown to be similar to histones from higher organisms [ 1 ] whereas a protein corresponding to histone Hl seems to be absent from this simple unicellular eukaryote [2]. Also the organization of yeast chromatin into nucleosomes closely resembles that of higher eukaryotes [ 11. Histones of other organisms have been found to be subject to various modifications. Phosphorylation, especially that of histones Hl and H3, occurs predominantly during chromosome condensation for mitosis in higher organisms [3] as well as in the slime mold, Physarum polycephalum [4]. Phosphorylation of histone H2a was suggested to be involved in interphase heterochromatin structure in a cell cycle-independent mamrer [5-71. Highly acetylated histone H4 was discovered specifically in transcriptionally active, DNase I-sensitive, chromatin [8-lo], its concentration is inversely correlated with the initiation of chromosome condensation in Physarum [II]. We asked whether such modifications, probably being devices for the modulation of DNA-histone interactions, also occurred in yeast and whether appearance of modified histone proteins varied during the vegetative cell cycle or during the sporulation process. We found incorporation of radioactive phosphate into yeast histone H2a and to a much smaller amount into H2b, but not into H3 and H4. The degree of incorporation did not change substantially during the vegetative growth cycle but increased considerably in cells preparing for sporulation. Radioactive sodium acetate was found to be incorporated into all core histone-bands. Analysis of unlabeled histones in acidurea gels, in which the acetylated variants of histones H4 and H3 can be separated, showed that these 2 histones in yeast exist as several subspecies acetylated to different degrees. Variations in the relative amounts of these subspecies were found during the mitotic cell cycle and less pronounced during the process of sporulation induction.

In vitro models for the identification and characterization of tumor-promoting and protective factors for colon carcinogenesis.

The present review aims to give a short overview of the existing cell culture models and their characteristics. While both the induction and the prevention of tumors can only be unequivocally demonstrated in vivo, in vitro models of colorectal cell growth are essential tools for the identification of candidate compounds and for the analysis of underlying mechanisms. Unfortunately normal and premalignant colorectal epithelial cells that represent the target cells of tumor promoters like desoxycholic acid or 1,2-diglyceride and could be used to investigate their cell biological effects are difficult to obtain. Cell line studies have mostly used carcinoma cell lines or rat small intestinal epithelial cells. Some normal immortalized cell lines have been established but are not widely used, and premalignant adenoma cell lines are extremely rare. However, those that do exist are useful both in mechanistic studies of cell growth and for the analysis of tumor promoters and chemoprotectors of colorectal carcinogenesis.

Histone synthesis during sporulation of yeast

Cells of the yeast, ~ffcc~ro~yces cerevisiae, can exist in three different states, the two haploid states, mating type ‘a or o, and the diploid state a/a! . Whereas a and cr cells are capable of mating with each other, a/a cells are able to sporulate and do not mate. These different abilities of cells are genetically governed by the mating type locus which is a fascinating example of a regulatory system functioning by DNA rearrangements as described by the cassette model [ 11. Diploid aJo cells can be looked upon as the ‘cell lineage’ which in contrast to cu and a cells (and also to laborato~~onst~cted o/o or ala cells) possess the ability to go, after receiving a signal ‘sta~ation’, through a meiotic division and to form haploid spores. We asked ourselves whether this process, which has some similarity to terminal cytodifferentiation of committed cells of a certain lineage in a higher organism, is reflected in a change of the histone pattern. Particularly, it was our aim to look for histone Hl , the presence or absence of which in vegetatively growing cells could not be unambiguously ascertained [2,3]. Although we did not obtain a more conclusive result concerning Hl , we found to our surprise that histone synthesis is uncoupled from premeiotic DNA replication and takes place in the early part of the sporulating process. This is in contrast to the situation in vegetatively growing yeast, in which histone synthesis [3] as well as the appearance of histone messenger RNAs (C. Viehauser, E. Wintersberger, unpublished) are tightly coupled to DNA replication during the. S phase of the mitotic cell cycle.

Retinoids inhibit protein Kinase C‐dependent transduction of 1,2‐diglyceride signals in human colonic tumor cells

1,2-Diglycerides with long-chain fatty acid residues related to nutritional fat (LCDGs) specifically affect growth and urokinase secretion in human colonic tumor cells, but not in normal mucosa. This allows them to advance and enhance carcinogenesis in the colon and rectum. SW480 colon carcinoma cells are LCDG sensitive in the same way as primary colonic tumor cells and have therefore been used as a model system to study the mechanism of LCDG action and to search for inhibitors of tumor development in the colon. Using this model system, we have shown that the effects of LCDGs are transmitted by protein kinase C and abolished by downregulation of the enzyme. Retinol, retinoic acid, and beta-carotene in nanomolar concentrations inhibit LCDG-induced growth and urokinase secretion and block stimulation of protein kinase C. Although retinol and retinoic acid at higher concentrations also display stimulatory activity, beta-carotene does not. At 100 nM, a concentration that can easily be reached in the plasma of humans, beta-carotene reduces LCDG-induced urokinase secretion about 50%. Inasmuch as beta-carotene does not have side effects due to intrinsic activities and storage effects, beta-carotene and foods rich in carotenes could be useful in the prevention of colorectal cancer.