Printing Ink Wastewater Treatment Using Hydrodynamic Cavitation and Coagulants/Flocculants.

Raw printing ink wastewater (PIW) was treated with various inorganic coagulants and organic flocculants (anionic and cationic polyacrylamides). These processes were also examined as post treatment step following hydrodynamic cavitation. Treatment effectiveness was assessed through color, chemical oxygen demand (COD) and total suspended solids (TSS) removal. The addition of 4500 mg L-1 polyaluminum chloride coagulant in undiluted PIW (COD: 17000 mg L-1) resulted in 99% color removal, 96% COD and TSS removal, after settling for 2 h. The addition of 10 mg L-1 of anionic polyacrylamides in the sample reduced settling time to only 5 min, with concomitant 96-98% removal efficiency. The addition of a 4 min hydrodynamic cavitation pretreatment step reduced coagulant addition by 33%, for the treatment of undiluted PIW (with 10 mg L-1 anionic polyacrylamide), while removals were ranged between 96 and 98%. Economic analysis for the undiluted PIW showed that costs were reduced by ca. 20% with the hydrodynamic cavitation pretreatment step. Moreover, sludge characterization showed the presence of maghemite, aluminum chloride and potassium aluminum silicate. Finally, toxicity tests revealed a significant attenuation of the toxic potential of undiluted PIW, thus indicating the enhanced efficiency of the proposed combined process (hydrodynamic cavitation and coagulation/flocculation).

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

ß-catenin negatively regulates expression of the prostaglandin transporter PGT in the normal intestinal epithelium and colorectal tumour cells: a role in the chemopreventive efficacy of aspirin?

BACKGROUND: Levels of the pro-tumorigenic prostaglandin PGE(2) are increased in colorectal cancer, previously attributed to increased synthesis through COX-2 upregulation and, more recently, to decreased catabolism. The functionally linked genes 15-prostaglandin dehydrogenase (15-PGDH) and the prostaglandin transporter PGT co-operate in prostaglandin degradation and are downregulated in colorectal cancer. We previously reported repression of 15-PGDH expression by the Wnt/ß-catenin pathway, commonly deregulated during early colorectal neoplasia. Here we asked whether ß-catenin also regulates PGT expression. METHODS: The effect of ß-catenin deletion in vivo was addressed by PGT immunostaining of ß-catenin(-/lox)-villin-cre-ERT2 mouse tissue. The effect of siRNA-mediated ß-catenin knockdown and dnTCF4 induction in vitro was addressed by semi-quantitative and quantitative real-time RT-PCR and immunoblotting. RESULTS: This study shows for the first time that deletion of ß-catenin in murine intestinal epithelium in vivo upregulates PGT protein, especially in the crypt epithelium. Furthermore, ß-catenin knockdown in vitro increases PGT expression in both colorectal adenoma- and carcinoma-derived cell lines, as does dnTCF4 induction in LS174T cells. CONCLUSIONS: These data suggest that ß-catenin employs a two-pronged approach to inhibiting prostaglandin turnover during colorectal neoplasia by repressing PGT expression in addition to 15-PGDH. Furthermore, our data highlight a potential mechanism that may contribute to the non-selective NSAID aspirin's chemopreventive efficacy.

The actin-bundling protein fascin is overexpressed in colorectal adenomas and promotes motility in adenoma cells in vitro.

BACKGROUND: Fascin is overexpressed in many cancers, including colorectal, but its role in the malignant transformation of benign colorectal adenomas is unclear. METHODS: Immunohistochemical analysis of fascin expression was carried out in resected human colorectal adenoma specimens. The effects of forced overexpression of fascin on adenoma cell motility were also analysed. RESULTS: We show fascin overexpression in adenomas increasing with tumour size, histological type, and degree of dysplasia and increased cell motility in adenoma cell lines following fascin transfection. CONCLUSION: These data suggest an important role for fascin in the malignant progression of colorectal tumours.

Insulin-like growth factor binding protein 3 (IGFBP-3) potentiates TRAIL-induced apoptosis of human colorectal carcinoma cells through inhibition of NF-kappaB.

There is growing evidence that the insulin-like growth factor-binding protein 3 (IGFBP-3) can have IGF-independent effects on cell growth. However, despite the fact that IGFBP-3 has been reported to be both antiproliferative and proapoptotic, the molecular mechanisms underlying the action of IGFBP-3 have not been elucidated. We report that although addition of IGFBP-3 (either synthetic or secreted protein) had no effect on cell survival, IGFBP-3 (100 ng/ml) significantly enhanced TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death in colonic carcinoma-derived cell lines (20-30% depending on cell line), whereas it had no effect on the survival of the TRAIL-resistant adenoma-derived cells. Both addition of IGFBP-3 protein to cell cultures or enforced expression of IGFBP-3 in the HT29 carcinoma cell line inhibited nuclear factor kappa B (NF-kappaB) activation in response to the induction of apoptosis by TRAIL. We propose that IGFBP-3 is a non-toxic NF-kappaB inhibitor, which could be used as an adjuvant in the treatment of colon cancer.

VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

Inhibition of COX-2 with NS-398 decreases colon cancer cell motility through blocking epidermal growth factor receptor transactivation: possibilities for combination therapy.

UNLABELLED: The use of non-steroidal anti-inflammatory drugs has proved of great interest in the prevention and treatment of colorectal cancer, although their precise mechanisms of action remain unclear. Overexpression of cyclooxygenase-2 (COX-2) and subsequent prostaglandin production promote metastasis and have been shown to increase cell motility in vitro. OBJECTIVE: We have aimed to elucidate whether specific inhibition of COX-2 with NS-398 (NS-398 is a selective inhibitor of COX-2) would be able to inhibit motility of colorectal cancer cells and whether this was modulated through epidermal growth factor receptor (EGFR) transactivation. MATERIALS AND METHODS: A transwell filter assay was used to study cell motility. Expression of COX-2, EGFR phosphorylation and prostaglandin E(2) (PGE(2)) receptors were assessed by Western blot analysis and reverse transcriptase-polymerase chain reaction. PGE(2) concentrations after NS-398 treatment were estimated by enzyme immunoassay. RESULTS: Treatment with NS-398 significantly reduced PGE(2) levels and reduced cell migration in the HT29 and HCA7 colorectal carcinoma cell lines and this effect was rescued by addition of PGE(2). Furthermore, specific inhibition of COX-2 with NS-398 reduced EGFR phosphorylation in colorectal cancer cells. Direct inhibition of EGFR activity with AG1478 reduced PGE(2)-stimulated motility, clearly demonstrating that PGE(2 )acts via the EGFR-signalling pathway. The novel combination of NS-398 and AG1478 dramatically reduced migration of colorectal cancer cells. CONCLUSION: The data presented indicate that the use of NS-398 in chemoprevention and adjuvant therapy for colorectal cancer may work in part, through the inhibition of cell motility. Furthermore, our data suggest that the combined use of non-steroidal anti-inflammatory drugs with EGFR antagonists could be explored further for future use in the clinic.

Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts.

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.

Membrane filtration of olive mill wastewater and exploitation of its fractions.

Olive mill wastewater (OMW) produced from small units scattered in rural areas of Southern Europe is a major source of pollution of surface and subsurface water. In the present work, a treatment scheme based on physical separation methods is presented. The investigation was carried out using a pilot-plant unit equipped with ultrafiltration, nanofiltration, and reverse osmosis membranes. Approximately 80% of the total volume of wastewater treated by the membrane units was sufficiently cleaned to meet the standards for irrigation water. The concentrated fractions collected in the treatment concentrates were characterized by high organic load and high content of phenolic compounds. The concentrates were tested in hydroponic systems to examine their toxicity towards undesired herbs. The calculations of the cost of the overall process showed that fixed and operational costs could be recovered from the exploitation of OMW byproducts as water for irrigation and/or as bioherbicides.

Neoplastic transformation of a human colonic epithelial cell line: in vitro evidence for the adenoma to carcinoma sequence.

The purpose of this study was to establish an in vitro model for tumor progression in colorectal carcinogenesis, by transforming the premalignant human colonic PC/AA adenoma cell line to the malignant phenotype. A rare clonogenic variant AA/C1 [colony-forming efficiency (CFE) on plastic of 1.05%] was isolated from the diploid PC/AA adenoma cell line (C. Paraskeva, S. Finerty, and S. Powell, Int. J. Cancer, 41: 908-912, 1988). AA/C1 was aneuploid and when treated with 1 mM sodium butyrate for 14 days gave rise to the AA/C1/SB cell line which had an increased CFE on plastic (6.13%) although the cells remained anchorage dependent and nontumorigenic. After exposure of these AA/C1/SB cells to the carcinogen N-methyl-N'-nitro-N'-nitrosoguanidine an anchorage-independent cell line was isolated (AA/C1/SB10). On continuous in vitro passage, the CFE in agarose of AA/C1/SB10 has increased to 17.3% and the cells have become tumorigenic producing adenocarcinomas in athymic nude mice. AA/C1, AA/C1/SB, and AA/C1/SB10 cell lines have common chromosomal abnormalities including a pericentric inversion of chromosome 1 with deletion of part of the short arm and monosomy for chromosome 18. This in vitro progression provides the first reported experimental evidence for the adenoma to carcinoma sequence in the human colon, and the cytogenetic evidence suggests that it is relevant to in vivo carcinogenesis.

Differential growth inhibition by the aspirin metabolite salicylate in human colorectal tumor cell lines: enhanced apoptosis in carcinoma and in vitro-transformed adenoma relative to adenoma relative to adenoma cell lines.

Regular aspirin intake may reduce the risk of colorectal cancer by 50%. However, the mechanism of this chemopreventive effect is not known. The effect of the aspirin metabolite salicylate on the growth of human colorectal tumor cell lines was determined. Salicylate showed dose-dependent inhibitory effects on all of the cell lines (IC50, 1.65 +/- 0.36 to 7.38 +/- 1.08 mM), yet carcinoma and in vitro-transformed adenoma cell lines were more sensitive than adenoma cell lines. Salicylate caused all cell lines to accumulate in G0-G1 and induced apoptosis in carcinoma and in vitro-transformed adenoma cell lines but not in all adenoma cell lines. In those adenoma lines that did show salicylate-induced apoptosis, the extent was considerably less than that in the more transformed cell lines. The ability of salicylate to induce cell cycle arrest and apoptosis and, in particular, the increased sensitivity of cells at later stages of neoplastic progression may be mechanistically important in the chemopreventive action of aspirin toward colorectal cancer.

Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy.

Vitamin D3 is believed to reduce the risk of colon cancer, and serum levels inversely correlate with colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of colon cancer cells. The vitamin D analogue, EB1089, is currently under clinical trial in a variety of cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agents. 1alpha,25-Dihydroxyvitamin D3 and EB1089 induced p53-independent apoptosis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the adenoma cell lines. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the carcinoma cell line HT29 was relatively resistant to apoptosis induced by EB1089 compared with 1alpha,25-dihydroxyvitamin D3, EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 was potentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was not essential for apoptosis. Levels of the proapoptotic protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinoma cells suggests that this or other vitamin D analogues may prove clinically effective for the treatment of colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.

Specific cytogenetic abnormalities in two new human colorectal adenoma-derived epithelial cell lines.

Two new epithelial cell lines from sporadic human colorectal adenomas designated S/AN and S/RG are reported. S/AN was from a villous adenoma and S/RG from a tubular adenoma. Both cell lines have extended growth capacities in vitro reaching passages 18 and 15, respectively, so far and show no signs of senescence. S/AN and S/RG have retained in vitro the ability to form mucin-producing goblet-like cells. Every cell of S/AN has a deletion on the short arm of chromosome 1 and one normal copy of chromosome 1. S/AN is also monosomic for chromosome 18. The majority of cells of S/RG only have one normal copy of chromosomes 6, 7, 14, 17, 18, and 22. S/RG also has several marker chromosomes. Although aneuploid S/AN and S/RG are nontumorigenic in athymic nude mice, these cytogenetic abnormalities are insufficient for the fully tumorigenic phenotype. The common abnormality for S/AN and S/RG is monosomy for chromosome 18, indicating that this is a central and important step in colorectal carcinogenesis. Our cytogenetic analysis of the adenoma cell lines suggests at least two possible routes by which premalignant colonic cells can develop and progress to malignancy. S/RG, unlike most other adenoma cell lines, is clonogenic. Aneuploidy, clonogenicity, and extended in vitro growth capacity may therefore be useful in vitro markers for adenoma cell lines with a relatively high malignant potential.

Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells.

We investigated the expression of insulin-like growth factor binding protein 3 (IGFBP-3) in normal human colonic epithelium and whether IGFBP-3 is involved in the induction of apoptosis in colonic epithelial cells. A gradient of IGFBP-3 protein expression was observed within the normal colonic crypt, and increased IGFBP-3 expression was coincident with the region of increased differentiation and apoptosis. Treatment of human colonic tumor cell lines with IGFBP-3 alone was shown to have no effect on growth. However, an increase in p53-dependent apoptosis was observed in the presence of 100 ng/ml IGFBP-3 24 h after the induction of DNA damage by gamma-irradiation. These results suggest that IGFBP-3 enhances the p53-dependent apoptotic response of colorectal cells to DNA damage.

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis.

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.

An acidic environment leads to p53 dependent induction of apoptosis in human adenoma and carcinoma cell lines: implications for clonal selection during colorectal carcinogenesis.

As tumours are known to acidify their microenvironment and fluctuations in lumenal pH have been reported in a number of colonic disease conditions, we investigated whether loss of p53 function, commonly associated with the adenoma to carcinoma transition in human colorectal epithelium, was implicated in the cellular response to changes in extracellular pH. Human colonic adenoma and carcinoma derived cell lines were incubated at an initial pH range of 5.5-8.0 and the attached cell yield and apoptotic cell yield determined after 4 days. Exposure of all cell lines to an acidic growth environment was associated with a G1 arrest, down regulation of the retinoblastoma protein (pRb) protein and switch to the hypophosphorylated form of the protein, and increased expression of the p21 protein. However, induction of apoptosis, associated with increased p53 protein expression but not with changes in Bcl-2 expression, was only detected in the adenoma derived BH/C1 and AA/C1 cell lines which express wild type p53 activity. Furthermore, this induction of apoptosis was inhibited in the transfected cell line AA/273p53/B, in which the wild type p53 function has been abrogated. These results suggest that acidification of the microenvironment would provide a selective growth advantage for cells that have lost wild type p53 function, leading to clonal expansion of aberrant cell populations.

Gamma-radiation-induced apoptosis in human colorectal adenoma and carcinoma cell lines can occur in the absence of wild type p53.

The tumour suppressor gene p53 codes for a transcription factor which is thought to play a critical role in the induction of G1 cell cycle arrest and programmed cell death (apoptosis) following DNA damage by ionizing radiation. The aim of this investigation was to determine whether a p53 independent radiation-induced apoptosis pathway exists in human colon epithelial cell lines. This report describes the induction, by gamma-radiation, of apoptosis in the colorectal adenoma cell line S/RG/C2, and in the colorectal carcinoma cell line PC/JW, both of which lack wild type p53. In addition, flow cytometry revealed that both cell lines failed to arrest in G1 after radiation. Thus, although loss of wild type p53 may abrogate G1 arrest, radiation-induced apoptosis can still occur in human colonic tumour cell lines through a p53 independent mechanism.

Differential sensitivity of human colonic adenoma and carcinoma cells to transforming growth factor beta (TGF-beta): conversion of an adenoma cell line to a tumorigenic phenotype is accompanied by a reduced response to the inhibitory effects of TGF-beta.

The growth of three non-tumorigenic human colonic adenoma cell lines, designated AA/C1, RG/C2 and RR/C1, was inhibited by low concentrations of transforming growth factor beta (TGF-beta) (0.05-0.5 ng ml-1). However, the growth of five human colon cancer cell lines under identical conditions was resistant to high concentrations of TGF-beta (2-10 ng ml-1). This is the first report of well-characterized premalignant human colonic cells showing sensitivity to TGF-beta. The TGF-beta-sensitive adenoma cell line AA/C1 was derived from a relatively large adenoma with a K-ras gene mutation and represents a relatively late-stage adenoma, indicating that loss of response to TGF-beta occurs at a relatively late stage in colorectal carcinogenesis and that the presence of a ras gene mutation does not necessarily confer resistance to TGF-beta. Of further interest, the RG/CZ cell line has a p53 mutation showing that p53 mutations do not necessarily lead to TGF-B insensitivity. Furthermore, in this paper we show that the conversion of the AA/C1 adenoma cell line to a tumorigenic phenotype [Williams et al., (1990) Cancer Res., 50, 4724] is accompanied by a reduced response to the growth-inhibitory effects of TGF-beta up to 10 ng ml-1. Reduced responsiveness to the inhibitory effects of TGF-beta may be an important event in the loss of growth control in colorectal carcinogenesis.